Fish health assessment is essential for maintaining sustainable aquatic ecosystems and ensuring the well-being of wild and farmed fish populations. Hematological parameters are crucial indicators of fish health, with poikilocytosis emerging as a fundamental marker with significant diagnostic value. Poikilocytosis refers to abnormally shaped erythrocytes in bloodstream, reflecting underlying physiological and pathological conditions. Poikilocytosis can occur in various fish species and can be influenced by environmental stressors, infectious agents, nutritional deficiencies, and exposure to pollutants. Morphological alterations in erythrocytes, such as acanthocytes, echinocytes, dacrocytes, schistocytes, spherocytes, and codocytes are common poikilocytes in fish. Understanding the relationship between poikilocytosis and fish health has important implications for disease diagnosis, monitoring, surveillance, and management. By quantifying poikilocytic changes, researchers and veterinarians can differentiate normal variations from pathological conditions, facilitating targeted interventions and treatment strategies. While most studies have focused on heavy metal toxicity, stressors, nutritional deficiencies, pollutants, and therapeutics, the etiological induction of poikilocytosis in fish health has been overlooked. Nonetheless, poikilocytosis remains a valuable biomarker for assessing fish health and their environment. This review highlights piscine poikilocytosis as a significant fish hematological biomarker and its importance in understanding their health and culture conditions.
INTRODUCTION
Fish, vital for ecosystems and human nutrition, face health challenges from diseases and environmental stressors [1-3]. Hematological biomarkers, notably poikilocytosis (abnormal erythrocyte shapes), are essential in fish health assessment, reflecting factors like age, sex, and reproductive efficiency [4-6]. Fish erythrocytes, critical for energy production, undergo morphological alterations before external signs, making poikilocytosis an early biomarker [4].
Poikilocytes, abnormally shaped erythrocytes, exhibit various forms like flat, elongated, teardrop, or crescent-shaped, indicating underlying disease pathology [7, 8]. Referred to as shape-shifting erythrocytes, poikilocytosis can result from internal damage, external stress, or genetic abnormalities [9, 10]. Fish poikilocytosis is linked to conditions such as viral infections, anemia, and oxidative stress, making it a valuable biomarker in fish health assessment [7, 11, 12]. Its usefulness has gained attention in aquaculture and fisheries management [2, 5].
Piscine poikilocytosis is induced by foreign particles, heavy metal toxicity, pharmaceuticals, and antibiotics, leading to altered erythrocyte morphology and anemic conditions [3, 5, 13-17]. Dietary florfenicol and emamectin benzoate cause erythro-morphological alterations in healthy Oreochromis niloticus juveniles [2, 3, 11, 12]. Poikilocytes have diverse characteristics; not all distorted erythrocytes imply poikilocytosis [18]. An accurate diagnosis of acquired poikilocytosis is vital, highlighting its suitability as a fish health indicator [18]. This review explores poikilocytosis in fish, covering its definition, classification, inducers, association with fish health conditions, and comparisons with other erythrocytic indices, offering insights into its potential applications in aquaculture and fisheries management.
DISCOVERY OF FISH POIKILOCYTOSIS
The term “poikilocyte” is derived from the Greek word “poikilos” meaning varied. Clinical poikilocytosis was first observed by [19], but its discovery in fish occurred later. The earliest documented report of “poikilocytosis” in teleosts was by Emmel [20] in his study on Phalloceros caudimaculatus. Emmel suggested that nonnucleated discs might originate from erythrocytes expelling their entire nucleus, considering poikilocytosis a contributing factor. Fox [21] observed imminent poikilocytosis with distorted nuclei in Terrapin spp. infected with tail disease, establishing a connection between erythrocyte morphology and pathological causes. Oria [22] found that schistocytes were occasionally formed when erythrocytes eliminated their nucleus through lysis, rhexis, or karyocytoplasmic disintegration. Smith [23] observed poikilocytic erythrocytes in coho salmon fed a folic acid-deficient diet, suggesting nutritional deficiencies play a role in these anomalies. Gill and Pant [24] reported mercury-induced poikilocytosis in Barbus conchonius, followed by subsequent studies on chromium [25] and zinc [26]. Heavy metal toxicity is a well-documented cause of poikilocytosis. Hoffman and Lommel [27] documented poikilocytosis in Salmo gairdneri following field exposure to proliferative kidney disease, indicating its link to anemia. Subsequent studies explored erythrocyte morphology in stressed fish, linking poikilocytosis to pesticides, insecticides, drugs, irradiation, and malnutrition (acquired poikilocytosis).
CAUSES OF POIKILOCYTOSIS
Anthropogenic activities, including pollution, malnutrition resulting from protein-rich diets in aquaculture, and the use of insecticides and pesticides, lead to the induction of poikilocytosis in fish [28, 29]. Microplastics in aquatic environments are a significant contributor to poikilocyte production [12, 30, 31]. These factors can cause cyto-genotoxicity, leading to erythro-membrane mutations persisting across generations [32]. High stocking environments and climate change also trigger poikilocytosis in fish [14, 33]. For a comprehensive understanding, Table 1 lists reported causes of poikilocytosis in aquaculture, highlighting the multifaceted nature of erythrocyte morpho-anomalies in fish.
TABLE 1.
An exhaustive compilation enumerating the underlying factors leading to the occurrence of hereditary and acquired poikilocytosis in aquaculture.
Cause
Mechanism of action
Reference
Hereditary poikilocytosis
Hemoglobin (Hb) anomalies
Mutations in the genes that encode hemoglobin subunits can lead to its structural changes, affecting its stability or oxygen-carrying capacity.
Homeoviscosal adaptation system can intercede by changing the fatty acid composition of the erythro-membrane.
Induces DNA damage through the release of DNase from lysosomes and the denaturation or thermal inactivation of enzymes involved in DNA repair leading to nuclear anomalies
Dysregulation in several transcripts encoding putative transcription factors linked with erythrocyte apoptosis
Increased dietary lipids provokes an increased erythrocyte surface area and membrane expansion as they interact with the erythro-membrane lipid bilayer
The binding of hemoglobin to the erythro-membrane enhances rigidity and diminishes porosity, while a decrease in cellular ATP levels promotes oxygen affinity by facilitating the unbound state of hemoglobin, consequently contributing to increased membrane fluidity
ATP depletion can disrupt the synthesis and turnover of membrane lipids, leading to changes in the lipid composition of erythro-membranes, hence reducing membrane stability
ATP depletion impairs ATP-dependent ion pumps like Na+/K+ ATPase, leading to altered ion gradients, sodium influx, cell swelling, and membrane distortion in poikilocytosis
Poikilocytosis can result from inherited genetic disorders, with excessive anthropogenic pressure in aquaculture potentially leading to hereditary poikilocytosis due to erythro-membrane mutations. In zebrafish (Danio rerio), a hereditary form of poikilocytosis known as ovalocytosis has been identified, characterized by oval-shaped erythrocytes instead of discoid shape, caused by mutations in the SPTB gene encoding β-spectrin protein [53, 54]. Zebrafish research has revealed various dyserythropoiesis manifestations, shedding light on specific gene mutations and their impact on erythrocyte morphology [54, 55]. For a comprehensive overview of acquired poikilocytosis in different fish species, refer to Table 2.
TABLE 2.
A comprehensive tabulation of documented poikilocytes in different species based on their cause of formation.
a
Poikilocytes mentioned are acnthocyte (Ac), echinocyte (Ec), drepanocyte (Drc), spherocyte (Spc), codocyte (Cc), schistocyte (Shc), dacrocyte (Dac), elliptocyte (Elc), degmacyte (Dec), and stomatocyte (Stc).
b
Organic compounds cited in this table are mainly phenols and their derivatives.
c
Environmental factors include pollution and contaminants from mill effluents.
Anemia can both result from and contribute to poikilocytosis [51, 149]. During anemia, the bone marrow compensates for the decreased red blood cell count by rapid production, leading to the formation of abnormally shaped erythrocytes or poikilocytes in the bloodstream [51]. Studies suggest that stress, hypoxia, and reduced ATP levels can depress erythropoiesis and lead to the mobilization of immature erythrocytes, causing their transformation and lysis in circulation, particularly under high doses of short-term exposure or lower doses of subchronic exposure. Oxidative stress also contributes to increased erythrolysis, resembling apoptosis and characterized by cell shrinkage, membrane blebbing, and phospholipid scrambling [39].
SPLEEN'S ROLE IN PISCINE POIKILOCYTOSIS
The spleen, acting as the principal site of erythropoiesis and a key reservoir for erythrocytes in various fish species, including elasmobranchs, holocephalans, and specific teleosts, captures blood-borne substances and eliminates deteriorating erythrocytes from circulation [150]. Elevated erythropoiesis in response to impaired erythrocyte function and removal of damaged cells via the spleen results in increased abnormally shaped erythrocytes [49]. Excessive blood flow through the spleen can hinder microcirculation, causing blockages [150]. Disruptions in protein–protein interactions within the fish erythro-membrane skeleton, occurring during filtration in the spleen, can lead to defective structural proteins and cytoskeletal fragility, inducing poikilocytosis [45]. These disruptions compromise the structure and function of the erythro-membrane [44]. Despite these challenges, fish tend to minimize erythrocyte fragmentation, allowing for the recovery of abnormal morphologies [44].
TYPES OF POIKILOCYTOSIS IN FISH
Various types of poikilocytes in fish have been extensively documented by researchers [5, 30, 44, 51]. It is crucial to note that fish erythrocytes have a nucleus, distinguishing them from mammalian erythrocytes. Therefore, shape abnormalities in piscine poikilocytes should not be directly compared to their mammalian counterparts. While some similarities exist, changes in fish poikilocytes may differ from those in humans or other mammals. For instance, codocytes, also known as target cells, exhibit a halo around the center in mammalian erythrocytes, whereas in fish erythrocytes, the halo is a hypochromic region around the nucleus [50]. Figure 1 provides a detailed comparison of different poikilocytes in fish and their mammalian counterparts.
Detailed illustration of diverse poikilocytes in teleosts, showcasting their distinct shape and their mammalian equivalents in comparison to normal piscine erythrocytes. Fish names having asterisks (*) are unpublished results and have only been added as a clear image. Spherocyte (S, Sp): [4, 47, 93, 101, 113, 125, 151]. Stomatocyte (S): [44, 47, 93, 105]. Degmacyte (Bi): [4, 129]. Elliptocyte (EL, El): [30, 47, 49, 87, 93, 126, 129, 130, 140, 152]. Dacrocyte (Da, Tr): [4, 47, 77, 87, 96, 129, 140, 152, 153]. Schistocyte (Sch, Sh): [5, 69, 77, 130, 154, 155]. Codocyte (C): [44, 93]. Drepanocyte (Sk, Skc, Cr): [77, 93, 130, 156]. Echinocyte (E, Ec, EC): [101, 116, 125, 129, 132, 140, 152, 157]. Acanthocyte (A, Ac): [70, 101].
Acanthocyte
Acanthocytes, characterized by irregularly spaced, variably sized spicules or projections on erythrocyte surfaces, are known as “spur cells” [29]. These spicules are believed to result from the expansion of the outer red cell membrane layer [4]. Focal changes in the sub-membrane cytoskeleton may predispose mammalian acanthocyte formation, with alterations in the Band 3 protein, responsible for maintaining the connection between the erythro-membrane and the sub-membrane cytoskeleton, being a possible factor [8]. Changes in the phosphorylation status of Band 3 have also been observed [158].
In higher vertebrates, acanthocyte formation can occur when the erythro-membrane contains an excess of cholesterol compared to phospholipids [159]. Changes in membrane lipids can be attributed to elevated blood cholesterol content or abnormal plasma lipoprotein composition due to faulty diets. Similar mechanisms of acanthocyte formation are documented in poikilothermic reptiles and amphibians, suggesting the presence of comparable pathways in fish [160]. Defective repair of oxidant-damaged erythrocyte phospholipids, particularly the acylation of lysophospholipids, has been reported [160].
Acanthocytes are susceptible to splenic trapping and destruction due to their abnormal morphology, leading to anemia [58]. It is worth noting that the “spiky” appearance of erythrocytes can also be an artifact caused by ethylene diamine tetra-acetic acid (EDTA) if there is a delay of more than 6 h between storage and smear preparation, emphasizing the importance of examining fresh peripheral blood smears for accuracy. Membrane fluidity, influenced by cholesterol, structural protein, and phospholipid proportions in the erythro-membrane, determines erythrocyte morphology. Acanthocytosis has been associated with liver dysfunction [29], suggesting a possible link between drug administration, organ damage, and poikilocytosis. In fish, acanthocytes have been observed during hemolytic, microcytic, and normocytic anemia induced by heavy metal intoxication, paper mill effluents, pharmaceutical application, pesticide application, and many more [50, 75, 96, 100], but their pathological significance has not been studied yet. Acanthocytes have been observed in every reported case of poikilocytosis in fish (Table 2). Increased proteolytic activity in the erythro-membrane, possibly due to a circulating factor, might contribute to acanthocyte formation.
Echinocyte
Echinocytes, also known as burr cells, derive their name from the Greek word meaning “sea urchin” due to their shell-like appearance. These cells exhibit a reversible alteration, meaning that their shape can change based on various factors, including the cell's environment, pH levels, metabolic state, and exposure to certain chemicals [125]. Echinocytes display rounded and evenly spaced projections surrounding the cells, accompanied by a central pallor. It is crucial to differentiate them from acanthocytes, which have irregularly spaced thorn-like projections and minimal or no central pallor.
In mammalian blood smears, crenation of erythrocytes is often caused by factors such as excess EDTA resulting from under-filled collection tubes, slow drying of the blood smear, exposure to a humid environment, or an alkaline pH due to the glass slides [8]. The formation of echinocytes is associated with an increase in the surface area of the outer lipid monolayer compared to the inner monolayer of erythrocytes. This transformation occurs in the presence of fatty acids, lysophospholipids, and certain drugs that are preferentially distributed in the outer half of the lipid bilayer [47, 157]. Dehydration of erythrocytes, increased pH, elevated intracellular calcium levels, and depletion of erythrocyte ATP can also lead to the formation of echinocytes [47].
Echinocytic cells have been observed in blood smears of Carassius carassius, indicating the presence of echinocytogenic substances in the aquatic environment, affecting membrane fluidity and rheological properties of the cell [157]. Echinocyte formation has been documented in fish exposed to gamma radiation, attributed to mechanisms such as erythrocyte dehydration, expansion of the outer membrane, and cellular energy depletion, inhibiting the ATP-dependent sodium/potassium pump and leading to echinocyte formation [109, 110]. Fish exposed to pesticides and insecticides exhibit abnormalities in erythrocytes via dehydration, inhibiting erythrocytic carbonic anhydrase activity, which is responsible for maintaining acid–base metabolism and osmoregulation [161]. Reports on echinocytes in aquaculture are abundant (Table 2), indicating their significance in fish health assessments.
Drepanocyte
Drepanocytes, also known as sickle-shaped erythrocytes, exhibit an elongated, crescent, or sickle-like morphology [4]. While commonly associated with sickle cell anemia in reptiles and amphibians [160], drepanocytes can also occur in fish. It has been observed that alkalosis, characterized by an increase in blood pH, can trigger drepanocyte formation [45] in veterinary pathology. Few reports are available on the occurrence of drepanocytes in fish (Table 2). However, drepanocytosis in fish is primarily linked to changes in hemoglobin due to inheritance rather than induced by external factors [160]. The understanding of drepanocytosis in fish is limited to hereditary anemic conditions, and further research is needed to elucidate the underlying mechanisms of acquired drepanocytosis.
Spherocyte
Spherocytes are abnormal erythrocytes with a spherical shape, appearing smaller and denser than regular red blood cells [104]. They lack the typical central pallor seen in normal erythrocytes, making their identification challenging [160]. While spherocytes may appear smaller on blood smears, they do not necessarily impact the mean cell volume (MCV). In fisheries, spherocytes are associated with severe hemolysis and genetic mutations in various erythro-membrane proteins [10, 125]. These mutations lead to membrane dysmorphisms, decreased surface area, reduced flexibility, and increased fragility, causing spherocytes to form. These abnormal cells are more susceptible to spleen capture and premature destruction, and severe cases may lead to reticulocytosis [10].
Codocyte
Target cells, also known as codocytes or leptocytes, are characterized by a central lump of hemoglobinized cytoplasm within the normal central pallor, giving them a “bullseye” appearance [44, 93]. This shape is due to an increased surface-to-volume ratio, allowing these cells to maintain deformability similar to normal cells and resist osmotic lysis [47]. In higher vertebrates, such as dogs and cats, codocytes are a normal feature in anemia and iron deficiency anemias due to excess membrane in young erythrocytes [160, 162]. In fish, target cells have been linked to heavy metal toxicity and environmental pollution, where an imbalance in intracellular volume results in a disproportionately smaller cell, leading to redundant membrane folding and the formation of a central core surrounded by a red peripheral ring and a pale halo [93]. Heavy metal toxicity in the liver reduces membrane cholesterol, weakening its tensile strength and causing codocyte formation [47]. Uneven hemoglobin distribution within the cell can also distort the surface area-to-volume ratio, leading to target cell formation [47]. It is important to note that mammalian codocytes may occur artifactually in high-humidity environments during blood smear preparation, a possibility in aquaculture settings [8]. Target cells in fish blood smears offer valuable insights into certain pathological conditions, indicating issues related to membrane composition and hemoglobin production [47].
Schistocyte
Schistocytes are fragmented erythrocytes commonly seen in iron deficiency anemia in reptiles [160]. In fish, their presence likely stems from the inherent mechanical fragility of red blood cells rather than vascular changes [69]. Schistocytes can occur in acquired and inherited red blood cell disorders, reflecting significant abnormalities in cell shape and size [163]. While being rarely present, studies have indicated the occurrence of schistocytes in fish, particularly under the influence of irradiation and gamma rays [71]. Fish with higher levels of unsaturated fatty acids in their membranes were found to be more susceptible to lipid peroxidation and erythro-membrane damage following gamma radiation [164]. These findings underscore the need for further research in this area.
Dacrocyte
Teardrop cells, or dacrocytes, are associated with thermal stress, heavy metal toxicity, irradiation, neoplasms, splenic abnormalities, oxidative stress, vitamin B12 and iron deficiencies, and hemolytic anemia in fish [5, 50, 77, 82, 85, 107, 165]. True dacrocytes exhibit blunted tips and point in different directions, while artifact teardrop cells have sharply pointed ends in the same direction [8]. Teardrop cell formation likely involves distortion as erythrocytes pass through the spleen's sinusoids [165]. Cyto-genotoxicity and UVA radiation exposure can cause dacrocytosis in fish by inducing apoptosis and morphological alterations in erythrocytes, leading to oxidative injury, cytoskeleton, and nucleus damage [72, 85, 166].
Elliptocyte
Elliptocytosis in fish is characterized by defects in the erythrocyte cytoskeletal network, primarily involving spectrin dimers or spectrin-actin-protein 4.1 junctional complexes [54]. Hereditary elliptocytosis, associated with mutations in genes encoding erythro-membrane proteins such as α-spectrin, ß-spectrin, and glycophorin C/Gerbich blood groups, causes defects in the horizontal cytoskeletal network [54]. Acquired elliptocytosis in fish, documented more frequently than hereditary cases, results in the formation of classical elliptocytes with parallel sides and a long axis more than twice the length of the short axis, as well as pencil cells characterized by a long axis more than three times the length of the short axis, commonly observed in severe anemic conditions [59, 64]. The elliptical shape emerges gradually due to mechanical stress in the circulation, caused by defects in erythro-membrane proteins, leading to increased mechanical weakness and membrane fragility [167]. While hereditary elliptocytosis has been reported in higher aquatic vertebrates [168], it has not been documented in fish, although mutations in genes encoding α-spectrin, ß-spectrin, or protein 4.1R can be postulated as potential causes [168].
Degmacyte
Bite cells, or degmacytes, are characteristic features found in diseases marked by the denaturation and precipitation of hemoglobin. In these conditions, hemoglobin precipitates within the spleen, leading to the removal of the affected membrane-hemoglobin segment, resulting in the distinct “bite” shape [8, 169]. Bite cells are commonly associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency, where the decrease in G6PD enzyme concentration as erythrocytes age can lead to hemolysis. Additionally, oxidative stress induced by drugs such as phenazopyridine, nitrofurantoin, sulfa agents, and infections can contribute to the formation of bite cells [170, 171].
Stomatocyte
Stomatocytes are a type of red blood cell characterized by a slit-like pallor area, resembling a “fish mouth” [100]. They can be observed in hereditary stomatocytosis as well as in certain acquired conditions related to liver damage due to pharmaceutical toxicity [44, 100, 105], with numerous cases documented in fish (Table 2). Stomatocytes result from genetic defects in membrane proteins, leading to increased ion permeability and fluid “leakiness”. These cells exhibit abnormal rigidity and poor deformability, contributing to rapid destruction. It is important to note that the slit-like appearance observed in dried films can be partially an artifact caused by decreased pH or exposure to certain compounds, emphasizing the need for fresh blood smears for accurate poikilocyte diagnosis [8].
Hereditary poikilocyte
Hereditary poikilocytosis in fish, particularly focusing on erythro-membrane and cytoskeletal gene mutations, has been investigated using zebrafish models [10, 54, 172]. Studies have primarily explored mutations in genes such as slc4a, which encodes a protein crucial for erythro-membrane stability and attachment to the cytoskeleton [10]. Mutations in slc4a destabilize the erythro-membrane, leading to hereditary spherocytosis [10]. Additionally, disruptions in proteins like β-spectrin and ankyrin, which are essential for maintaining erythro-membrane stability and deformability, have been linked to hereditary poikilocytosis in fish [44, 54, 64, 173]. These genetic defects in key proteins contribute to the loss of erythrocyte elasticity and deformability, representing fundamental causes of hereditary poikilocytosis in aquaculture, consequently impacting crucial physiological aspects in fish. These changes might disrupt blood viscosity, impair oxygen transport, and diminish immune tolerance. The repercussions might extend beyond affecting the growth and development of fish progeny; they can also result in reduced reproductive success. This holds particular significance in fish hatcheries where maintaining the quality of hatchlings is imperative. Therefore, vigilant monitoring and a thorough understanding of the physiological implications arising from hereditary erythrocyte deformities become indispensable. This awareness is vital for the effective management and conservation of fish populations, whether in aquaculture settings or natural environments, and thus, further research is warranted.
PATHOLOGICAL, NUTRITIONAL, AND TOXICOLOGICAL INFLUENCE ON POIKILOCYTOSIS
Erythro-morphological alterations serve as bioindicators of environmental, pathogenic, or anthropogenic influences. These anomalies have been observed in freshwater fish exposed to hypoxia, stressed fish, splenic abnormalities, and several hematopoietic disorders like anemia (Table 2). However, this can vary based on factors like nutritional deficiencies, toxicosis from substances like heavy metals and pesticides, and malnutrition. All the documented reports of piscine poikilocytosis till date have been grouped under several influencing factors and expressed as a heat map (Figure 2). Fish exposed to peroxide-producing yeasts or unhealthy diets show pronounced poikilocytosis. In the reverse scenario, recovery periods differ among species and stimuli [33, 45, 49, 69, 75, 120, 125, 153, 174-177]. Erythrocyte morphology in fish can be altered due to various pathological conditions induced by etiological agents. Non-regenerative anemia, including hemolytic, hemorrhagic, and hypochromic anemia, results from inflammatory diseases, toxins, and renal or splenic disorders, leading to poikilocytosis [44, 75]. Hemolytic anemia in fish is caused by hemolysin-producing bacteria, viral infections, and hemoparasites such as Trypanosoma spp. and helminthes, with common pathogens like Aeromonas spp., Vibrio anguillarum, and Pseudomonas spp. contributing to the condition [7, 9]. Induced anemia affects hematopoietic tissues, especially the spleen, resulting in abnormal erythrocyte morphology. Protistan parasites form cellular inclusions in erythrocytes, leading to the presence of dacrocytes and schistocytes in the bloodstream. Elevated eosinophil counts in fish indicate inflammatory responses associated with antigenic stimulation or parasitic infections, potentially contributing to poikilocytosis. While bacterial and parasitic causes of induced poikilocytosis are documented, the association of viral infections with poikilocytosis in fish remains underexplored [7]. A detailed list of the pathological influence on piscine poikilocytosis are listed in Table 3.
Heat map depiction of all documented cases of poikilocytosis in aquaculture linked to various influencing factors (pathological, nutritional, toxicological, and external environmental factors) expressed as percentage.
TABLE 3.
A comprehensive overview of the documentation of different poikilocytes in various pathological conditions.
a
Miscellaneous erythrocyte morphological alterations which included changes in erythrocyte morphometry but were not documented as a particular poikilocyte.
INFLUENCE OF EXTERNAL ENVIRONMENTAL CONDITIONS ON POIKILOCYTOSIS
Fish experience poikilocytosis as a response to various environmental stressors, including hypoxia, toxins, and temperature changes [44, 75]. Climate change and rising sea temperatures, alongside factors such as ocean acidification, oxygen levels, salinity changes, habitat loss, and altered migration patterns, contribute to the vulnerability of aquatic ecosystems, leading to poikilocytosis and hematological alterations in marine organisms [187]. Climate-induced stress brings about changes in lipid composition, leading to alterations in erythro-membrane properties and disruptions in protein–lipid interactions. These modifications impact the dynamics of cytoskeletal proteins [41, 188]. Furthermore, genotoxicity, cytotoxicity, and severe poikilocytosis in fish are triggered by UV and gamma irradiation, as well as aquatic pollution stemming from sources like detergents, tanneries, slaughterhouses, and microplastics [28, 29, 37, 45]. These environmental stressors pose significant threats to aquatic biomes and the overall health of aquatic organisms. A schematic diagram of all the external environmental factors contributing to piscine poikilocytosis are documented in Figure 3.
Schematic representation of all the major non-pathological environmental factors influencing the induction of piscine poikilocytosis.
Environmental factors significantly impact fish erythrocytes. Seasonal changes, driven by water temperature fluctuations, can alter erythrocyte numbers and hemoglobin content [146]. Warm waters have been linked to poikilocytosis in species like Rutilus rutilus [189]. Hypoxic environments are associated with erythro-nuclear anomalies, including dacrocytes, in fish [87, 88, 190]. Starvation affects erythrocyte turnover, potentially leading to anemic conditions [6, 191].
CHANGES IN ERYTHROCYTIC INDICES DURING POIKILOCYTOSIS
Fish erythrocyte parameters vary due to factors like diet, size, age, sex, temperature, and environment [6, 7]. Adverse conditions, including toxicity and nutritional deficiencies, often lead to reduced erythrocyte count (ERC), hemoglobin concentration (Hb), hematocrit (Hct), and mean corpuscular volume (MCV), causing anemia. Poikilocytosis, a rare correlation with hematologic parameters, serves as an essential indicator of fish health and stress responses [192]. Specific poikilocytes, like dacrocytes, are linked to conditions such as nutritional deficiency and jaundice, leading to decreased erythrocytic parameters. Severe toxicity exposure can cause various types of anemia, with macrocytic anemia showing increased MCV and mean corpuscular hemoglobin concentration, while other types exhibit decreased indices [6]. Although erythro-morphological alterations are associated with changes in erythrocytic indices [31, 153], definitive correlations between specific poikilocytes and induced hematological anomalies are lacking. Poikilocytosis analysis, complemented by erythrocytic index estimation, is crucial. Other indices like erythrocyte sedimentation rate, red blood cell distribution width (RDW), and erythropoietic precursor indices could potentially enhance poikilocytosis analysis, but comprehensive data are limited.
IMPORTANCE OF VISUALIZING POIKILOCYTOSIS IN ENSURING FISH HEALTH IN AQUACULTURE
Fish erythrocytes, vital for oxygen transport and maintaining balance, are often assessed through quantitative parameters, overlooking intricate morphology [45, 135, 193]. Physiological alterations in fish spleens, often accompanied by elliptocytes, dacrocytes, and schistocytes, are indicators of faulty splenic functions due to various factors, including drug toxicity and exposure to industrial effluents [2, 3, 5, 12, 194]. Detection of irregular erythrocyte shapes, especially poikilocytes, indicates anemic conditions, hematological disorders, nutritional deficiencies, or toxic exposures [195]. Specific poikilocytes like schistocytes and codocytes signify hemolytic anemia or liver disease, respectively [7, 196, 197], and their observance can be a signal for potential pathogenic involvement. In such instances, additional hematological tests should be conducted to remain vigilant for potential disease outbreaks. Under any poikilocytic circumstances, it is crucial to assess water quality parameters to ensure they are within optimal ranges, and subsequent actions may be necessary. Additionally, as spherocytosis and stomatocytosis can be attributed to drugs and synthetic chemicals, their usage should be promptly discontinued, and regular blood smearing should be monitored for signs of improvement. However, it is important to note that drug-induced poikilocytosis may occur during therapeutic treatments and should not be mistaken for other conditions. Degmacytes are rarely seen in minor cases of irradiation and thermal stress [129], making it challenging to establish correlations with specific environmental or pathological conditions. Poikilocytosis, along with erythro-morphological anomalies like polychromatophilia, lobopodia, and membrane internalization, serves as a sensitive biomarker indicating physiological responses to pollutants, pharmaceuticals, and pathogens in fish [7, 45]. In documenting poikilocytosis, beyond assessing heavy metal presence and maintaining optimal water quality, prompt monitoring of potential causes and associated behavioral anomalies (erratic swimming, gasping, anorexia, dark pigmentation, and lethargy) is essential to contain disease outbreaks and reduce mortality. Additionally, minimizing anthropogenic activities such as bathing, washing, waste dumping, and agricultural runoff is crucial. Regular weekly monitoring of feed quality is also recommended to prevent fungal growth. Routine blood smear examinations can detect anomalies early, enabling proactive measures and timely action, enhancing biosecurity protocols and ensuring the well-being of aquatic ecosystems [76]. Fish red blood cell morphology is more sensitive to certain toxic chemicals than basic hematology parameters, making poikilocytosis a valuable diagnostic tool even when traditional indices like Ht, total ERC, or Hb remain relatively stable [198]. Studies on hematological anomalies induced by microplastics and nanoparticles provide evidence of anemic conditions in fish following exposure to these stressors [30, 199]. Irradiation induces pronounced poikilocytosis, suggesting its potential as an early indicator of thermal stress in fish [77]. Fish, with their unique capacity to metabolize, concentrate, and store diverse pollutants, emerge as excellent subjects for discerning potential mutagenic and carcinogenic effects of contaminants in water. In laboratory conditions, the application of acute and prolonged doses of gamma radiation has been acknowledged for inducing piscine erythro-morphological alterations [112, 200, 201]. This phenomenon is characterized by the presence of echinocytes, microcytes, and anisochromatic erythrocytes in fish blood under such radiation conditions. These poikilocytes show promise as potential indicators of cytotoxic and genotoxic damage to peripheral erythrocytes in fish exposed to nuclear radiation, thereby emphasizing the significance of piscine poikilocytosis as an indicator of nuclear pollution. Extensive erythro-morphological changes have been documented in studies on captured fish from reservoirs and other water bodies within the Chernobyl Exclusion Zone, as reported by Gudkov et al. [202] and Pomortseva and Gudkov [200]. Consequently, hematological disorders offer a valuable means to assess the impact of nuclear pollution on marine and freshwater fish biota. It is posited that qualitative indexes of erythrocytes in the peripheral blood of fish prove more sensitive to long-term radiation impact rendering them suitable for conducting hematologic monitoring of radioactively contaminated water bodies or areas.
However, till date, in the case of any poikilocytes, making definitive deductions based solely on their presence becomes challenging and requires further research. Even the combination of different poikilocyte types may yield similar interpretations. Therefore, the observation of any poikilocyte should prompt subsequent hematological tests, providing a foundation for further nuanced deductions.
CURRENT RESEARCH HOT SPOTS, EMERGING TRENDS, AND ADVANCEMENTS IN THE STUDY OF POIKILOCYTOSIS IN AQUACULTURE
Despite advancements in medical technology, the analysis of erythrocyte morphology remains fundamental in hematological assessment, with a growing emphasis on poikilocytosis as a crucial biomarker for xenobiotic exposure and environmental quality [157, 194, 195]. Ongoing research is increasingly dedicated to exploring hematological changes induced by microplastics, and their contamination in aquatic environments has raised concerns in modern aquaculture [94, 203]. There is a belief that these changes may be irreversible or necessitate an extended recovery period, underscoring the need for further investigation. Beyond microplastic contamination, poikilocytosis has emerged as a pivotal outcome in pesticide and insecticide contamination in fish, highlighting cytotoxicity [69, 140, 177]. The escalating usage of such synthetic chemicals has compromised fish health, leading to extensive research on molecular mechanisms of erythrocyte genotoxicity [78, 204]. Increasing attention is directed toward hematological disorders as a primary diagnosis of etiological involvement, given that blood corpuscles typically show the first signs. Recent research on Heteropneustes fossilis infected with haemoparasites has underscored proliferative poikilocytosis, emphasizing the importance of understanding morphological changes in erythrocytes during diseases for fish health management and aquaculture practices [45, 205]. Additionally, the impact of nanoparticles on fish erythrocytes and their potential role in poikilocytosis, particularly in the context of nanotechnology in aquaculture, remains a promising but understudied area [83, 206]. As nanoparticles are considered the future of drug delivery and pathogen control due to their bactericidal properties, increasing research studies have explored the effects of different nanoparticles on fish erythrocytes, shedding light on erythro-morphological abnormalities in this context [207]. In most recent reports, Aliko et al. [207] described comprehensive erythro-morphological abnormalities in response to metallic nanoparticle exposure.
CHALLENGES IN ASSESSMENT OF PISCINE POIKILOCYTOSIS
Poikilocytosis occurs in response to stress-induced metabolic changes in fish [6]. However, diagnosing poikilocytosis, whether in fish or mammals, requires additional blood tests and expertise for accurate interpretation. It is challenging to use a single poikilocyte type as a diagnostic criterion. Clear definitions of different poikilocytes are lacking, hindering reproducibility and application in aquaculture. Challenges arise from the diversity in fish erythrocyte shapes among species and age-groups, as well as differences between teleosts and elasmobranchs [195]. Interpretation of poikilocytic responses is complex due to the diverse factors influencing erythro-morphological alterations in fish exposed to xenobiotics. Current erythrocyte profiling technologies are limited in assessing shape, necessitating labor-intensive approaches. However, digital smear images and automated technologies show promise pending species standardization and validation [14, 195]. Research increasingly focuses on erythrocytes' sensitivity to xenobiotics, emphasizing their role as efficient bioindicators of fish health in aquatic environments [208]. Poikilocytosis offers valuable insights into fish health, reflecting xenobiotic-induced damage and toxicity in aquatic ecosystems.
CONCLUSION
Fish hematology, although trailing behind other vertebrates, offers valuable insights into disease processes in teleosts and elasmobranchs. The term “erythro-morphological anomalies” is more suitable for aquaculture than “poikilocyte,” which has historically been used in clinical contexts. Poikilocytosis in fish signifies a significant hematological phenomenon with implications for health and disease diagnosis. This review, a pioneering effort, emphasizes the lesser-known poikilocytosis in fish. Fish pathologists and nutritionists are encouraged to analyze samples, benefiting individual animals and contributing to reference data. Abnormal erythrocyte morphology provides insights into fish pathophysiology, indicating conditions like parasitic infections, anemia, deficiencies, inflammatory diseases, and toxic exposures. Specific poikilocytic characteristics inform evaluations and treatment strategies. Erythrocyte sensitivity to environmental pollution makes their morphological assessment a reliable bioindicator. Poikilocytosis is a diagnostic tool for organ condition, anthropogenic influence, and climate change effects, serving as a biomarker for fish health and culture conditions. It not only provides diagnostic information but also delves into the complex realm of fish hematological health, aiding advancements in fish health management and conservation practices. Ongoing research enhances our understanding of poikilocytosis, its molecular mechanisms, and facilitates progress in fish health management and conservation.
Authors would like to acknowledge funding from the Indian Council of Agricultural Research (ICAR), Government of India, New Delhi, under All India Network Project on Fish Health (Grant F. No. CIBA/AINP-FH/2015-16) dated 02.06.2015.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interests that could potentially harm the publication of the manuscript.
ETHICS STATEMENT
The current article used extensive literature from different authors and resources for comprehensive review preparation, and henceforth, no animal ethics were harmed or are required to be stated.
Data sharing is not applicable to this article as no new data were created or analyzed in this study.
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