Hemolymph Cells Research Articles

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be ‘aroused’ to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.

Nucleoprotein of a Rice Rhabdovirus Serves as the Effector to Attenuate Hemolymph Melanization and Facilitate Viral Persistent Propagation in its Leafhopper Vector.

Melanization in the hemolymph of arthropods is a conserved defense strategy against infection by invading pathogens. Numerous plant viruses are persistently transmitted by insect vectors, and must overcome hemolymph melanization. Here, we determine that the plant rhabdovirus rice stripe mosaic virus (RSMV) has evolved to evade the antiviral melanization response in the hemolymph in leafhopepr vectors. After virions enter vector hemolymph cells, viral nucleoprotein N is initially synthesized and directly interacts with prophenoloxidase (PPO), a core component of the melanization pathway and this process strongly activates the expression of PPO. Furthermore, such interaction could effectively inhibit the proteolytic cleavage of the zymogen PPO to active phenoloxidase (PO), finally suppressing hemolymph melanization. The knockdown of PPO expression or treatment with the PO inhibitor also suppresses hemolymph melanization and causes viral excessive accumulation, finally causing a high insect mortality rate. Consistent with this function, microinjection of N into leafhopper vectors attenuates melanization and promotes viral infection. These findings demonstrate that RSMV N serves as the effector to attenuate hemolymph melanization and facilitate viral persistent propagation in its insect vector. Our findings provide the insights in the understanding of ongoing arms race of insect immunity defense and viral counter-defense.

Anesthesia with Tricaine Methanesulfonate (MS222) and Propofol and Its Use for Computed Tomography of Red Swamp Crayfish (Procambarus clarkii).

Crayfish (Decapoda: Astacoidea and Parastacoidea) are among the few animals that have stem cells in hemolymph, with the capacity to continuously produce differentiated neuronal structures throughout life. As the use of crayfish and other invertebrates increases in biomedical research, we must develop laboratory standards and guidelines for performing clinical procedures. This manuscript presents introductory protocols for anesthesia in crayfish during diagnostic imaging. Five anesthetic protocols were evaluated: immersion in buffered tricaine methanesulfonate (MS222; 50 mg/L); immersion in buffered MS222 (150 mg/L); immersion in propofol (65 mg/L); injection of propofol (50 mg/kg); and injection of propofol (100 mg/kg) into the ventral surface of an abdominal somite. MS222 immersion (50 and 150 mg/L) had no observable effect on crayfish. After an extended period of time, immersion in propofol (65 mg/L) created a sedative effect suitable for short-term handling. Propofol injection (50 mg/kg) into the ventral surface of an abdominal somite created an effective plane of anesthesia without adverse effects during or after recovery. Propofol injection at 100 mg/kg had adverse effects and is not recommended for use in crayfish. CT imaging was performed successfully as proof of concept for handling anesthetized crayfish. These findings provide initial data for the anesthetization of crayfish used in research settings.

Tatlı su midyesinin (Unio delicatus) hemosit parametreleri üzerine Cyfluthrinin etkilerinin belirlenmesi

The overuse of pesticides has been increasing since the 20th century. Depending on this use, non-target organisms are also affected apart from target organisms. Cyfluthrin is a synthetic pyrethroid pesticide used in agriculture, domestic and veterinary medicine against insects. It may also affect non-target aquatic organisms as a result of mixing with aquatic ecosystems. This study was aimed to investigate the effect of cyfluthrin on freshwater mussels, one of the aquatic invertebrate species, with hemocyte parameters. Total hemolymph counts, hemolymph cell morphology, and differential hemocyte counts were performed from hemolymph taken from mussels exposed to different cyfluthrin doses exposures for 24 and 48 hours. Compared to the control group, the total hemocyte counts of the experimental groups were found to increase in 24h and decrease in 48h significantly (p<0.05). In the examination of hemocyte morphologies, granular, semi granular, and hyalinocyte cells were observed. Similar values of differential hemocyte counts were found both 24 and 48h exposure times. As a result, in aquatic toxicology studies, besides total hemocyte count, analysis of hemocyte morphologies and differential hemocyte counts are found to be good biomarkers.

Immune response in the larva of the dung beetle Phyllognathus excavatus against human blood cells as foreign bodies

The dung beetle Phyllognathus excavatus (Coleoptera: Scarabaeidae) is a widespread and common species throughout its distribution (North Africa, southern Europe, and Asia). The larvae remain most of their life in compost-rich soil feeding on organic matter. This report aimed to identify and describe the larval immune effector cells (hemocytes) types and to investigate their immunological responses. For this, hemolymph was collected to investigate the different circulating cells using both light and scanning electron microscopy. The Larval hemolymph cells were identified and hemocytes immune responses under-reacting with human blood cells was investigated. The hemogram parameters were analyzed in the larval hemolymph, mixture of larval hemolymph and human blood, and healthy adult man blood (as a reference). Experiments showed that granulocytes, plasmatocytes, and coagulocytes were involved in the first line in the lysis of human red blood cells. Results showed that the larvae present also a great number of platelets-like and lymphocytes-like. Besides, the majority of the human red blood cells were killed under the larval immune defense. Thus, the content of human red blood cells was reduced by 75 % when mixed with the larval hemolymph whose student test showed a significant decrease (α < 0.01). Our findings, validated through immunological responses induced by larvae hemocytes towards human bood cells, show that hemocytes from scarab larvae, involved in immune defense mechanisms, can defend against infections by foreign pathogens and offer an insight into the novel application for mammal diseases.

Effect of Extreme Heatwaves on the Mortality and Cellular Immune Responses of Purplish Bifurcate Mussel Mytilisepta virgata (Wiegmann, 1837) (=Septifer virgatus) in Indoor Mesocosm Experiments

In the rocky intertidal environment, the frequency and duration of heatwaves have increased over the last decade, possibly due to global climate change. Heatwaves often result in lethal or sub-lethal disturbances in benthic animals by changing their metabolic activities. In this study, we investigated the impacts of extreme heatwave stress on the hemocyte functions of Mytilisepta virgata and subsequent mortality to gain a better understanding of the potential causes and consequences of mass mortality events in this mussel during summer. We discriminated three types of hemocytes in the hemolymph, granulocytes, hyalinocytes, and blast-like cells, using flow cytometry and revealed that granulocytes were the major hemocyte involved in cellular defensive activities, such as phagocytosis and reactive oxygen species (ROS) production. For the experiment, mussels were exposed to a 40°C air temperature for 12 h per day over 5 days under laboratory conditions as a simulated semi-diurnal tidal cycle. Mortality began to occur within 3 days after beginning the experiment, and all mussels had died by the end of the experiment. Flow cytometry indicated that the mussels exposed to high air temperatures produced significantly more ROS than did the control mussels within 2 days after the onset of the experiment, which may have caused oxidative stress. Such high levels of ROS in the hemolymph increased DNA damage in hemocytes after 3 days of exposure and decreased the phagocytosis of hemocytes 4 days after the experiment began. The observed mortality and decline in immune capacity suggested that an extreme heat event occurring in the rocky intertidal ecosystem during summer could exert sublethal to lethal impacts on macrobenthic animals.

Wing hearts in four-winged Ultrabithorax-mutant flies-the role of Hox genes in wing heart specification.

Wings are probably the most advanced evolutionary novelty in insects. In the fruit fly Drosophila melanogaster, proper development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove hemolymph and apoptotic epidermal cells from the premature wings through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant flies that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later second and third thoracic segments. However, adult dipterian flies harbor only one pair of wings and only one pair of associated wing hearts in the second thoracic segment. Here we show that the specification of WHPs depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analyzed the development of wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment is homeotically transformed into a second thoracic segment resulting in a second pair of wings instead of the club-shaped halteres. We show that a second pair of functional wing hearts is formed in the transformed third thoracic segment and that all wing hearts originate from the wild-type population of wing heart progenitor cells.

Cell type diversity in scallop adductor muscles revealed by single-cell RNA-Seq

Studies on cell atlas in marine invertebrates provide a better understanding of cell types, stem cell maintenance, and lineages of cell differentiation. To investigate the molecular features of various cell types in molluscan muscles, we performed single-cell RNA sequencing (scRNA-seq) to map cell types in scallop adductor muscles. We uncovered the cell type-specific features of 20 cell clusters defined by the expression of multiple specific molecular markers. These cell clusters are mainly classified into four broad classes, including mesenchymal stem cells, muscle cells, neurons, and haemolymph cells. In particular, we identified a diverse repertoire of neurons in the striated adductor muscle, but not in the smooth muscle. We further reconstructed the cell differentiation events using all the cell clusters by single-cell pseudotemporal trajectories. By integrating dual BrdU-PCNA immunodetection, neuron-specific staining and electron microscopy observation, we showed the spatial distribution of mesenchymal stem cells and neurons in striated adductor muscle of scallops. The present findings will not only be useful to address the cell type-specific gene expression profiles in scallop muscles, but also provide valuable resources for cross-species comparison of marine organisms.

Drosophila suzukii Susceptibility to the Oral Administration of Bacillus thuringiensis, Xenorhabdus nematophila and Its Secondary Metabolites.

Simple SummaryIn recent decades, climate change and the international fruit trade have favored the movement of allochthonous species such as harmful insects into new geographic areas. The settlement of phytophagous insects and vectors in new areas, where potential predators are often lacking, has increased the use of chemical insecticides for their control. The intensive use of these substances represents a serious problem for ecosystems and human health; a possible alternative to chemical control is biological control, i.e., the use of biological insecticides that are compatible with the environment. The aim of our work was to further improve biological control methods for the management of the dipteran Spotted Wing Drosophila, an insect recently introduced in America and Europe, which can damage thin-skinned fruit crops. The methodologies applied are based on the combined use of different entomopathogens, i.e., bacteria, fungi, nematodes, etc., harmful for insects, with the purpose of increasing their effectiveness. The results obtained show that the combined use of two entomopathogenic bacteria increases both the lethality and rapidity of action. From an application viewpoint, studies like this are essential to identify new methods and bioinsecticides and, once transferred to the field, can be crucial to eliminate or, at least, reduce the use of chemicals.Drosophila suzukii, Spotted Wing Drosophila (SWD), is a serious economic issue for thin-skinned fruit farmers. The invasion of this dipteran is mainly counteracted by chemical control methods; however, it would be desirable to replace them with biological control. All assays were performed with Bacillus thuringiensis (Bt), Xenorhabdus nematophila (Xn), and Xn secretions, administered orally in single or combination, then larval lethality was assessed at different times. Gut damage caused by Bt and the influence on Xn into the hemocoelic cavity was also evaluated. In addition, the hemolymph cell population was analyzed after treatments. The data obtained show that the combined use of Bt plus Xn secretions on larvae, compared to single administration of bacteria, significantly improved the efficacy and reduced the time of treatments. The results confirm the destructive action of Bt on the gut of SWD larvae, and that Bt-induced alteration promotes the passage of Xn to the hemocoel cavity. Furthermore, hemocytes decrease after bioinsecticides treatments. Our study demonstrates that combining bioinsecticides can improve the efficacy of biocontrol and such combinations should be tested in greenhouse and in field in the near future.

Development of a VP28-pseudotyped baculovirus expression system for efficient gene transfer in penaeid shrimps

Both efficient gene transfer and expression tools and suitable reporter genes are lacking for penaeid shrimps and their isolated cells. In this study a VP28-pseudotyped insect baculovirus expression system: Bacmid-GUS/VP28, carrying a reporter gene of GUS (β-glucuronidase) and an exogenous envelope protein of VP28 from shrimp white spot syndrome virus (WSSV), has been successfully developed for the first time and well used for efficient gene transfer and expression in adult shrimps with an extremely high infection efficiency of up to 100%, but in a tissue-specific manner because it could only infect the adult tissues of gill, heart and intestine, but not the Oka organ and muscle under the tested infection doses. The aforementioned VP28-pseudotyped recombinant baculovirus of Bacmid-GUS/VP28 could also infect the primarily cultured hemolymph cells of shrimp (Metapenaeus ensiss) and produced strong GUS signals with a relatively low efficiency of 1.21%, but failed in the primary embryonic cells of shrimp (Penaeus japonicus), showing an obvious cell-specificity. A possible explanation for the above-mentioned low infection efficiency is the occurrence of mitosis-arrest in the infected shrimp cells. In contrast, no obvious GUS signals could be detected in any of the uninfected and wild type recombinant baculovirus (Bacmid-GUS)-infected tissues and cells of adult shrimps, confirming the suitability of GUS as a reporter gene and the great contribution of VP28 to the tissue- and cell- tropism of pseudotyped recombinant baculovirus (Bacmid-GUS/VP28) in penaeid shrimps. In a word, the successful development of VP28-pseudotyped recombinant baculovirus system will provide us a useful tool for efficient gene transfer and expression in adult shrimps as well as an assay system for the examination of the contribution of different WSSV envelope proteins to the viral tissue tropism.

Antifungal effects of Streptococcus mutans extract on Candida strains susceptible and resistant to fluconazole: An in vivo study.

Streptococcus mutans crude extract shows antifungal effects on clinical Candida strains susceptible and resistant to fluconazole in Galleria mellonella model.

Possible Roles of Hypotaurine and Thiotaurine in the Vesicomyid Clam Phreagena okutanii.

AbstractVesicomyid clams, which inhabit deep-sea hydrothermal vents and hydrocarbon seeps, are nutritionally dependent on symbiotic, chemoautotrophic bacteria that produce organic matter by using hydrogen sulfide. Vesicomyid clams absorb hydrogen sulfide from the foot and transport it in their hemolymph to symbionts in the gill. However, mechanisms to cope with hydrogen sulfide toxicity are not fully understood. Previous studies on vent-specific invertebrates, including bathymodiolin mussels, suggest that hypotaurine, a precursor of taurine, mitigates hydrogen sulfide toxicity by binding it to bisulfide ion, so as to synthesize thiotaurine. In this study, we cloned cDNAs from the vesicomyid clam Phreagena okutanii for the taurine transporter that transports hypotaurine into cells and for cysteine dioxygenase and cysteine-sulfinate decarboxylase, major enzymes involved in hypotaurine synthesis. Results of reverse-transcription polymerase chain reaction indicate that mRNAs of these three genes are most abundant in the foot, followed by the gill. However, hypotaurine and thiotaurine levels, measured by reverse-phase high-performance liquid chromatography, were low in the foot and high in the gill. In addition, thiotaurine was detected in hemolymph cells. Hypotaurine synthesized in the foot may be transported to the gill after binding to bisulfide ion, possibly by hemolymph cells.

AGE RELATED HEMOCYTE COMPOSITION IN AUTUMN GENERATION OF APIS MELLIFERA L.

AGE RELATED HEMOCYTE COMPOSITION IN AUTUMN GENERATION OF APIS MELLIFERA L.

Functional Characterization of the Pacific Oyster, Crassostrea gigas (Bivalvia: Ostreidae), Hemocytes Under Normoxia and Short-Term Hypoxia

Hemolymph cellular composition, morphology and functional properties of the Pacific oyster (Crassostrea gigas) hemocytes were studied. Three hemocyte types (agranulocytes, hyalinocytes and granulocytes) were described in hemolymph. The morphology of each type was characterized by light microscopy and flow cytometry. Agranular cells (agranulocytes and hyalinocytes) were the dominant type of cells in hemolymph; their number was 86.7±2.7% of total cell count. Under hypoxia the number of agranulocytes increased (37.4% for control group versus 95.3% for hypoxic probes), whereas granulocyte and hyalinocyte number decreased up to 3.9% and 0.7% in hypoxic specimens respectively. The spontaneous ROS production decreased in each hemocyte type after exposure to hypoxia. Low dissolved oxygen did not influence hemocyte proliferation and mortality level.

MORPHOMETRIC CHARACTERISTICS OF HEMOCYTES IN WORKER BEES APIS MELLIFERA L.

Introduction. Hemocytes of bees perform many functions. Cellular immunity is one of the most important among them. This part of the immune system provides wound healing, protection against parasites and pathogens. Research of hemocytes composition is important for the study of the cellular component of bee immunity under the influence of biotic and abiotic factors.Aim. The aim of our research was to study the hemocytes composition of worker bees Apis mellifera L. The experiment was performed during the autumn-winter period on worker honey bees.Methods. Insects were selected from the colonies zoned in Chernivtsi region. Then hemolymph was selected, smears were made, fixed and stained, and microscopy was performed. Longitudinal and transverse diameters were measured in the identified hemolymph cells and their nuclei.Results. The detected prohemocytes are the cells of the smallest size, of round or oval shape, with a compact nucleus stained in dark purple. The cytoplasm surrounds the nucleus with a thin layer. Longitudinal and transverse diameters of prohemocyte nuclei are 5.41 ± 0.12 and 4.99 ± 0.10 μm. Some of the detected plasmatocytes are oval-shaped, others are spindle-shaped. The nuclei of plasma cells, located mainly in the center of the cells, are stained in dark pink or pink-purple. Chromatin is condensed in the form of grains. The cytoplasm is transparent. It contains light pink or light purple small granules, sometimes it contains vacuoles. The average values of the longitudinal and transverse diameters of oval plasmatocytes are 12.02 ± 0.15 and 7.03 ± 0.12 μm and for their nuclei they are 7.64 ± 0.13 and 4.09 ± 0.11 μm. The average values of the longitudinal and transverse diameters of spindle-shaped plasmatocytes are 15.30 ± 0.21; 5.21 ± 0.16 μm, and for their nuclei they are 8.42 ± 0.08; 4.26 ± 0.07. The granulocytes have a large round or oval pink-purple nucleus. The dimensions of the nucleus vary considerably: the longitudinal diameter is from 10.85 to 19.85 μm, and the transverse diameter is from 8.24 to 19.63 μm. Longitudinal and transverse diameters of the 1st type permeabilized cells nuclei are 7.64 ± 0.26 мкм and 6.75 ± 0.24 μm, permeabilized cells of the 2nd type have an amoeboid shape with the longitudinal and transverse nucleus diameters 7.31 ± 0.21 and 6.56 ± 0.23 μm. Transitional form of cells are morphologically different from differentiated cells.Conclusion. Prohemocytes, oval and spindle-shaped plasmatocytes, granulocytes, permeabilized cells and transitional cell forms were detected in hemolymph of worker honey bees during the autumn-winter period. Key words: Apis mellifera; hemocytes; morphometric characteristic

Study of the proliferative activity of hemolymph cells in pulmonate molluscs

Hemocytes, the cell of the hemolymph, play a key role in the immune response of pulmonate molluscs to various pathogens including trematode infection. The number of hemocytes is known to increase after immunization but the mechanism of their multiplication remains debatable, proliferative capacity being the stumbling block. Some scientists consider that hemocytes may proliferate, while others think that their multiplication is only possible in special hematopoietic centres. In this work we studied the proliferative activity of hemocytes in five species of pulmonate molluscs: Biomphalaria glabrata, Planorbarius corneus, Planorbis planorbis, Lymnaea stagnalis and Succinea putris. ImageStream technique was used for the study of the hemocyte populations of these molluscan species for the first time. The hemocytes of all the studied species were represented by two main types, granular cells and hyalinocytes. Microscopic and flow-cytometric study of the hemocytes with the use of EdU revealed some EdU-positive cells. However, the analysis of the cell cycle of the hemocytes showed that the amount of DNA in these cells was not increased. Thus, it remains unclear whether the cells of the hemolymph retain the capacity to multiplication.

Differences in proteomic profile between two haemocyte types, granulocytes and hyalinocytes, of the flat oyster Ostrea edulis

Haemocytes play a dominant role in shellfish immunity, being considered the main defence effector cells in molluscs. These cells are known to be responsible for many functions, including chemotaxis, cellular recognition, attachment, aggregation, shell repair and nutrient transport and digestion. There are two basic cell types of bivalve haemocytes morphologically distinguishable, hyalinocytes and granulocytes; however, functional differences and specific abilities are poorly understood: granulocytes are believed to be more efficient in killing microorganisms, while hyalinocytes are thought to be more specialised in clotting and wound healing. A proteomic approach was implemented to find qualitative differences in the protein profile between granulocytes and hyalinocytes of the European flat oyster, Ostrea edulis, as a way to evaluate functional differences. Oyster haemolymph cells were differentially separated by Percoll® density gradient centrifugation. Granulocyte and hyalinocyte proteins were separated by 2D-PAGE and their protein profiles were analysed and compared with PD Quest software; the protein spots exclusive for each haemocyte type were excised from gels and analysed by MALDI-TOF/TOF with a combination of mass spectrometry (MS) and MS/MS for sequencing and protein identification. A total of 34 proteins were identified, 20 unique to granulocytes and 14 to hyalinocytes. The results suggested differences between the haemocyte types in signal transduction, apoptosis, oxidation reduction processes, cytoskeleton, phagocytosis and pathogen recognition. These results contribute to identify differential roles of each haemocyte type and to better understand the oyster immunity mechanisms, which should help to fight oyster diseases.

Analysis of Cell Cycle and Morphological and Functional Abnormalities of Mytilus galloprovincialis Lam., 1819 (Bivalvia) Hemocytes from Coastal Ecosystems near Sevastopol, Crimea

The Black Sea coastal waters near Sevastopol are used intensively for household and industrial purposes and are characterized by high human disturbance. To investigate the level of environmental toxic impact on the organism functional state, this study analyzes changes in hemolymph cell composition, morphology, DNA content, and cell cycle parameters of bivalve mollusk Mytilus galloprovincialis from six experimental sites. The mollusks from the mussel farm on Donuzlav Lake (Crimea) are the control group. Significant differences have been found between mussels from the control area and coastal waters near Sevastopol in main morphological and functional parameters such as cell size, granularity, and nucleus-cytoplasmic ratio. Mussels from the Sevastopol area are characterized by abnormalities of the hemocyte cell cycle manifested in aneuploidy and an increase in the intraindividual variability of genome size and proportion of cells with 4c DNA content up to 34.80–65.5%, which most likely reflects the cell transition to polyploidy.

Morphologic, cytometric and functional characterisation of Anadara kagoshimensis hemocytes

Circulating hemocytes of ark clam, Anadara kagoshimensis, were investigated using light microscopy and flow cytometry. Hemolymph contained 3 morphotype of cells, amebocytes, erythrocytes and intermediate type cells, which formed two distinct subpopulations on flow cytometric dot plots. Large cells (intermediate morphotype and erythrocytes) amounted 85.6 ± 2.8% total cells in hemolymph. Erythrocytes were hemoglobin-containing cells with up to 40 granules presented in the cytoplasm. All hemocyte types, observed in the ark clam hemolymph demonstrated equal capacity to spontaneous production of reactive oxygen species.

ГЕМОЦИТИ ІНТАКТНОЇ ТА ІНВАЗОВАНОЇ ТРЕМАТОДАМИ ЖАБУРНИЦІ КИТАЙСЬКОЇ (MOLLUSCA, UNIONIDAE, ANODONTINAE)

The hemolymph of Sinanodonta woodiana (Lea, 1834) consists of plasma and cells of four types: prohemocytes (cambial cells), macronucleocytes, basophilic and eosinophilic granulocytes. The three latter cell types derive from the cambial cells though mitosis. The cellular sizes are: prohemocytes 14.3±0.4, basophilic granulocytes 20.9±0.7, young eosinophilic granulocytes 23.8±0.6, older eosinophilic granulocytes 25.7±0.1, macronucleocytes 26.1±0.3. All cells and their nuclei are roundish. Nuclear chromatin is either fine-grained fairly evenly distributed in the karyoplasm (in basophilic granulocytes), or more or less grouped dark-colored small (2-5-6) chromatin blocks. NC-ratio is maximum in macronucleocytes (0.6±0.01). NC-ratio of prohemocytes is 0.4±0.01, that of basophilic granulocytes is 0.3±0.01. NC-ratio of eosinophilic granulocytes is 0.2±0.01. Eosinophilic cells prevail in hemolymph elements: the young eosinophilic granulocytes make up 25.1±0.4 % and the older eosinophilic cells are up to 27.9±0.6 % of all hemocytes. The major functions of hemolymph cells are transport and protection.Transport is particularly pronounced in basophilic granulocytes in the form of phagocytosis. The protective function of hemocytes of different categories is manifested in a different way. Thus, some of the basophilic granulocytes develop into nephrocytes, which accumulate numerous (11-23) vacuoles of yellow-green-brown color. The vacuoles are subsequently excreted by the mollusk through the kidneys. The protective function of eosinophilic granulocytes is realized as false agglutination (these hemocytes clog into lumps that close wounds), which helps preventing blood loss. Also, eosinophilic granulocytes merge into multinucleated plasmodium, accumulating in large quantities around various foreign bodies (parasites or fragments of destroyed tissues) and encapsulating them to isolate from host tissues. At Northern Black Sea Coast, Chinese pond mussel is a common intermediate host of the trematode Rhipidocotyle companula Dujardin, 1845.The trematode inhabits the mollusk’s gonads. The parasitic sporocysts and cercariae were found in 29 % of examined mollusk specimens. Infestations were weak (up to 10 % of gonads were affected) in 22.4 % of infected mollusks, moderate (10 to 50 % of gonads) in 70 % of contaminated mollusks. Only 7.6 % of infected pond mussels were hyperinfected (100 % of gonads were affected).Weak trematode infestation is accompanied by localized damage. The total number and volume of parasitic focal lesions are generally insignificant. Moderate infection, and especially a hyperinfection cause not only the lesions in the hostal biotope, but also the overall pathological process in mollusk hosts. Simultaneously, the prohemocytes and basophilic granulocytes as well as their nuclei reduce in size. The total number of prohemocytes also declines by 1.7 times. The greatest decrease in the nuclear-cytoplasmic ratio (by a factor of 1.5-2) is noted also for prohemocytes and basophilic granulocytes. The vacuolization of karyoplasm and cytoplasm, the basophilization of cytoplasm, the degenerative changes in hemocyte nuclei (karyopicnosis, karyorexis, karyolysis), and the total number of aging and dying hemocytes are directly related to the level of infection intensity.