Types Of Hemocytes Research Articles

Morphological and functional characterization of circulating hemocytes in Tribolium castaneum larvae.

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye-staining properties were used to characterize hemocyte types from T.castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2h), then increased over time (at 4 and 6h) and eventually returned to the naive state by 24h post-injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T.castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph invivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes invitro, forming capsules to encapsulate and melanize nickel-nitrilotriacetic acid (Ni-NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T.castaneum larvae, offering valuable insights into cell-mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T.castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T.castaneum hemocytes in combating pathogens.

Chlorantraniliprole Enhances Cellular Immunity in Larvae of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae).

The innate immunity of insects encompasses cellular and humoral defense mechanisms and constitutes the primary defense against invading microbial pathogens. Cellular immunity (phagocytosis, nodulation, and encapsulation) is primarily mediated by hemocytes. Plasmatocytes and granulocytes play an important role and require changes in the cytoskeletons of hemocytes. However, research investigating the immunological impacts of insecticides on the fall armyworm (FAW), Spodoptera frugiperda, remains scarce. Therefore, we conducted a study to investigate the effects of chlorantraniliprole exposure on cellular immunity in FAW larvae. Our findings revealed the presence of five types of hemocytes in the larvae: prohemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. The LD10, LD20, and LD30 of chlorantraniliprole affected both the morphology and total count of some hemocytes in the larvae. Moreover, larvae exposed to chlorantraniliprole showed increased phagocytosis, nodulation, and encapsulation. To determine the mechanism of the enhanced cellular immunity, we studied plasmatocytes in the spread state and the cytoskeleton in hemocytes. It was found that the spreading ratio of plasmatocytes and the areas of the cytoskeletons in hemocytes were increased after chlorantraniliprole treatment. These results suggest that exposure to chlorantraniliprole results in an enhanced immune response function in FAW larvae, which may be mediated by cytoskeletal changes and plasmatocyte spreading. Consequently, this study provides valuable insights into the cellular immune response of FAW larvae to insecticide exposure.

Hemolymph Parameters Are a Useful Tool for Assessing Bivalve Health and Water Quality

Bivalves play a key role in aquatic ecosystems and are a valuable commercial resource. The prosperity of these aquatic organisms depends mainly on the effectiveness of their immune defense, in which the hemolymph plays a central role. Hemolymph may be used as an effective non-lethal criterion of health. However, the predictive value of hemolymph analysis depends on the comparison between the obtained results and reference data from healthy individuals living in natural aquatic environments. We collected hemolymph from 15 commercially important species from wild populations at stations located in non-impacted and impacted water areas of the Sea of Japan. Of the 11 hemolymph parameters we analyzed, the total hemocyte count, percentage of hemocyte types, phagocytic activity, presence of reactive oxygen species, and protein concentration differed significantly between populations from non-impacted and impacted water areas. The most responsive species to pollution were Magallana gigas, Crenomytilus grayanus, Mizuhopecten yessoensis, and Mactra chinensis. This work is the first to examine a large number of commercially important species simultaneously. The results of this study are the basis for establishing the health status criteria of commercial bivalves for veterinary control in aquaculture and biomonitoring.

Longitudinal tracking of hemocyte populations in vivo indicates lineage relationships and supports neural progenitor identity in adult neurogenesis

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types — hyaline (HC), semigranular (SGC) and granular (GC) cells — with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

INFLUENCE OF ALUMINUM OXIDE NANOPARTICLES ON BIOLOGICAL FEATURES AND HOST HEMOCYTES OF Galleria mellonella L. (Lepidoptera: Pyralidae) WITH ITS ENDOPARASITOID Pimpla turionellae L. (Hymenoptera: Ichneumonidae)

Nanoparticles (NPs) are released directly or indirectly into nature with increased production and consumption, and their effects on insects, which occupy a large place in the ecosystem, are of interest. There is also interest in the potentially toxic effects of NPs applied to hive pests on parasitoids, or honey bees, and host-parasitoid relationships. The influence of aluminum oxide (Al2O3) NPs on the biological features of the hive pest Galleria mellonella, total counts of hemocyte, and hemocyte types; as well as on the biological features of the endoparasitoid Pimpla turionellae were investigated. The data obtained revealed that Al2O3 NPs caused a decrease in the larval, pupal, and adult development time of G. mellonella. The immature developmental time of P. turionellae was reduced. It was also demonstrated that Al2O3 NPs decreased the total counts of hemocytes in G. mellonella larvae; granulocyte, spherulocyte, oenocytoid, and prohemocyte counts decreased at all NP concentrations, while plasmatocyte counts increased. The data showed that Al2O3 NPs affected the biological properties of the hive pest model organism G. mellonella and indirectly affected its endoparasitoid P. turionellae. In addition, NPs showed a suppressive effect on cellular immune system responses, decreasing hemocyte counts. Our study results suggest that especially honey bees, honeycomb pests, and parasitoids may be negatively affected by NPs, which have increased in recent years as environmental pollutants, and that NPs may have insecticidal effects.

Analysis of X-ray irradiation effects on the mortality values and hemolymph immune cell composition of Apis mellifera and its parasite, Varroa destructor

Varroa destructor is one of the most destructive enemies of the honey bee, Apis mellifera all around the world. Several control methods are known to control V. destructor, but the efficacy of several alternative control methods remains unexplored. Irradiation can be one of these unknown solutions but before practical application, the effectiveness, and the physiological effects of ionizing radiation on the host and the parasite are waiting to be tested. Therefore, the objective of our study was to investigate the effects of different doses (15, 50, 100, and 150 Gy) of high-energy X-ray irradiation through mortality rates and hemocyte composition changes in A. mellifera workers and record the mortality rates of the parasite. The mortality rate was recorded during short-term (12, 24, and 48 h) and long-term periods (3, 6, 12, 18, and 24d).The sensitivity of the host and the parasite in case of the higher doses of radiation tested (50, 100, and 150 Gy) been demonstrated by total mortality of the host and 90 % of its parasite has been observed on the 18th day after the irradiation. V. destructor showed higher sensitivity (1.52-times higher than the adult honey bee workers) at the lowest dose (15 Gy). A. mellifera hemocytes were influenced significantly by radiation dosage and the elapsed time after treatment. The higher radiation doses increased plasmatocyte numbers in parallel with the decrease in prohemocyte numbers. On the contrary, the numbers of granulocytes and oencoytes increased in the treated samples, but the putative effects of the different dosages on the recorded number of these hemocyte types could not be statistically proven. In summary, based on the outcome of our study X-ray irradiation can be deemed an effective tool for controlling phoretic V. destructor. However, further research is needed to understand the physiological response of the affected organisms.

Cellular Immunity of Drosophila willistoni Reveals Novel Complexity in Insect Anti-Parasitoid Defense.

Coevolution of hosts and their parasites has shaped heterogeneity of effector hemocyte types, providing immune defense reactions with variable effectiveness. In this work, we characterize hemocytes of Drosophila willistoni, a species that has evolved a cellular immune system with extensive variation and a high degree of plasticity. Monoclonal antibodies were raised and used in indirect immunofluorescence experiments to characterize hemocyte subpopulations, follow their functional features and differentiation. Pagocytosis and parasitization assays were used to determine the functional characteristics of hemocyte types. Samples were visualized using confocal and epifluorescence microscopy. We identified a new multinucleated giant hemocyte (MGH) type, which differentiates in the course of the cellular immune response to parasitoids. These cells differentiate in the circulation through nuclear division and cell fusion, and can also be derived from the central hematopoietic organ, the lymph gland. They have a binary function as they take up bacteria by phagocytosis and are involved in the encapsulation and elimination of the parasitoid. Here, we show that, in response to large foreign particles, such as parasitoids, MGHs differentiate, have a binary function and contribute to a highly effective cellular immune response, similar to the foreign body giant cells of vertebrates.

Dissecting Combinational Mechanisms of Herbal Formula from a Transcriptome-based Multi-scale Network Pharmacology Model

Abstract Objective: Illumination of the integrative effects of herbs in a formula is a bottleneck that limits the development of traditional Chinese medicine (TCM). In the present study, we developed a transcriptome-based multi-scale network pharmacology model to explore the combined effects of different herbs. Materials and Methods: First, we curated gene signatures at different biological scales, from the molecular to higher tissue levels, including tissues, cells, pathological processes, biological processes, pathways, and targets. Second, using the Xiexin Tang (XXT) formula as an example, we collected transcriptomic data in response to the treatment of XXT or its three compositive herbs on Michigan cancer foundation7 cells. Third, we linked each herbal drug to different biological scales by calculating the correlation scores between herb-induced gene expression profiles and gene signatures. Finally, the combined mechanisms of the three constituent herbs in XXT were deciphered by comparing their multi-scale effects with those of the formula. Results: The results showed that although XXT or single herbs regulated a large number of signatures on each biological scale, the phenotypic effects of these herbal drugs are concentrated onto the “Blood” tissue, types of hemocytes, and hemorrhagic injury-related pathological processes. At the molecular level, these herbs consistently regulate processes such as the cell cycle and blood coagulation-related pathways, as well as protein targets related to the immunoinflammatory response and blood coagulation, such as proteinase-activated receptor 2, integrin beta-3, inhibitor of nuclear factor kappa-B kinase subunit beta, and coagulation factor XII. The analysis of the combinational modes demonstrated that different herbs can cooperate by acting on the same objects and/or regulating different objects in related functions, and cooperative behaviors change at different biological scales. Conclusions: Our model can dissect the combined effects of herbal formulae from a multi-scale perspective and should be beneficial for the development and exploitation of TCM.

Single-cell RNA-seq revealed heterogeneous responses and functional differentiation of hemocytes against white spot syndrome virus infection in Litopenaeus vannamei.

Current knowledge of shrimp hemocyte classification mainly comes from morphology, which hinder in-depth characterization of cell lineage development, functional differentiation, and different immune response of hemocyte types during pathogenic infections. Here, single-cell RNA sequencing was used for mapping hemocytes during white spot syndrome virus (WSSV) infection in Litopenaeus vannamei, identifying 16 cell clusters and evaluating their potential antiviral functional characteristics. We have described the dynamic balance between viral infestation and hemocyte immunity. And the functional differentiation of hemocytes under WSSV stimulation was further characterized. Our results provided a comprehensive transcriptional landscape and revealed the heterogeneous immune response in shrimp hemocytes during WSSV infection.

NO production of granulocytes associated with antibacterial immune response in Tegillarca granosa

Nitric oxide (NO) is a signaling molecule and immune effector produced by the nitric oxide synthases (NOS), which involved to various physiological processes of animals. In marine bivalves, hemocytes play important roles in antimicrobial innate immune response. Although hemocyte-derived NO has been detected in several bivalves, the immune function of hemocyte-derived NO is not well understood. Here, we investigated the antibacterial response of hemocyte-derived NO in the blood clam Tegillarca granosa. Two types of hemocytes including erythrocytes and granulocytes were isolated by Percoll density gradient centrifugation, their NO production and TgNOS expression level were analyzed. The results showed that NO was mainly produced in granulocytes and almost no detected in erythrocytes. The granulocytes showed significantly higher NO level and TgNOS expression level than the erythrocytes. And the TgNOS expression level was significantly increased in granulocytes after Vibro parahemolyticus challenge. In addition, the NO donor sodium nitroprusside (SNP) significantly increased the NO production of hemocytes to kill pathogenic bacteria. In summary, the results revealed that granulocytes-derived NO play vital roles in the antimicrobial immune response of the blood clam.

Antiviral properties of Penaeus monodon cyclophilin A in response to white spot syndrome virus infection in the black tiger shrimp

Cyclophilin A (CypA) or peptidylprolyl isomerase A, plays an important role in protein folding, trafficking, environmental stress, cell signaling and apoptosis etc. In shrimp, the mRNA expression level of PmCypA was stimulated by LPS. In this study, all three types of shrimp hemocytes: hyaline cell, granulocyte and semi-granulocyte expressed the PmCypA protein. The mRNA expression level of PmCypA was found to be up-regulate to four-fold in white spot syndrome virus (WSSV) infected hemocytes at 48 h. Interestingly, PmCypA protein was only detected extracellularly in shrimp plasma at 24 h post WSSV infection. To find out the function of extracellular PmCypA, the recombinant PmCypA (rPmCypA) was produced and administrated in shrimp primary hemocyte cell culture to observe the antiviral properties. In rPmCypA-administrated hemocyte cell culture, the mRNA transcripts of WSSV intermediate early gene, ie1 and early gene, wsv477 were significantly decreased but not that of late gene, vp28. To explore the antiviral mechanism of PmCypA, the expression of PmCypA in shrimp hemocytes was silenced and the expression of immune-related genes were investigated. Surprisingly, the suppression of PmCypA affected other gene expression, decreasing of penaeidin, PmHHAP and PmCaspase and increasing of C-type lectin. Our results suggested that the PmCypA might plays important role in anti-WSSV via apoptosis pathway. Further studies of PmCypA underlying antiviral mechanism are underway to show its biological function in shrimp immunity.

Characterization of the effects of Chinese peony (Radix paeoniae Alba) and its active ingredients on the hematopoietic function of the Chinese mitten crab (Eriocheir sinensis)

Hematopoietic tissue (HPT) is crucial for the proliferation and release of newly produced hemocytes during infection in crustaceans. Very few studies have reported how herbs affected the hematopoietic function of crustaceans. Radix paeoniae Alba is a candidate immunopotentiator herb with the potential to be a blood-tonifying drug. In this study, the effects of Radix paeoniae Alba and its monomers (paeoniflorin, albiflorin, and paeonol) on HPT and hemocytes of Eriocheir sinensis were evaluated. After feeding them commercial diets containing different ingredients for three weeks, the HPT development was determined via morphological observation and hematopoietic tissue body index (HTBI) calculation. The count and classification of hemocytes were evaluated using the hemocytometer and flow cytometer. The expression of hematopoietic factor astakine, transglutaminase (TGase), and marker genes for different types of hemocytes (KPI and SOD) and undifferentiated HPT cells (PCNA) were detected using real-time quantitative PCR (qRT-PCR) assay. The results indicated that the HTBI in the Radix Paeoniae Alba, paeoniflorin, and albiflorin groups increased by 114.96%, 111.42%, and 112.50%, which were higher than the control group. The Radix Paeoniae Alba, paeoniflorin, albiflorin, and paeonol groups had higher proportions of ovoid lobules than the control group, especially in the Radix Paeoniae Alba group. The total hemocyte count (THC) of the crab in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups were 8.12, 9.69, and 8.57×106 ind/mL, respectively, which were higher than the control group. Furthermore, all experimental groups showed a higher proportion of granulocytes than the control group, with increased astakine protein being simultaneously detected in the HPT. The qRT-PCR results showed a relatively higher KPI expression in all the experimental groups, and also higher SOD mRNA expression in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups, as compared with the control group. Therefore, these results implied the potential effect of Radix Paeoniae Alba and its monomers on the hematopoietic function of E. sinensis. However, due to the complex ingredient composition in Radix Paeoniae Alba and their different activities in the hematopoietic process, further research needs to be conducted in the future.

Hemocyte morphology of worker subcastes of the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Hemocytes are cells present in the hemolymph of insects that play a role in combating invasive pathogens, ensuring defense by the immune system in these organisms. While the types of hemocytes are well known in some insect representatives, data on these cells in Hymenoptera are limited to certain bees and wasps, with little information available for ants. Among ants, the genus Atta has environmental and economic importance, forming highly organized colonies consisting of the queen and workers, with the latter subdivided into subcastes: gardeners, waste removers, foragers, and soldiers, which are exposed to different pathogens. This study describes the morphology of hemocytes in the worker subcastes of Atta sexdens rubropilosa. Hemolymph samples from the ant were submitted to light, confocal, and scanning electron microscopy analyses. Five types of hemocytes were identified in the hemolymph of all ant subcastes, including prohemocytes, oenocytoids, spherulocytes, plasmatocytes, and granulocytes. They exhibited nuclei with a predominance of decondensed chromatin. The granulocytes were the most abundant cell type in the subcastes, followed by prohemocytes, plasmatocytes, oenocytoids, and spherulocytes. Phagocytosis assays reveal that plasmatocytes and granulocytes are the main phagocytic cells in all castes evaluated. This study fills an important gap in understanding the immune response in this ant species.

The plant toxin 4-methylsulfinylbutyl isothiocyanate decreases herbivore performance and modulates cellular and humoral immunity.

Insect herbivores frequently encounter plant defense molecules, but the physiological and ecological consequences for their immune systems are not fully understood. The majority of studies attempting to relate levels of plant defensive chemistry to herbivore immune responses have used natural population or species-level variation in plant defensive chemistry. Yet, this potentially confounds the effects of plant defense chemistry with other potential plant trait differences that may affect the expression of herbivore immunity. We used an artificial diet containing known quantities of a plant toxin (4-methylsulfinylbutyl isothiocyanate; 4MSOB-ITC or ITC, a breakdown product of the glucosinolate glucoraphanin upon herbivory) to explicitly explore the effects of a plant toxin on the cellular and humoral immune responses of the generalist herbivore Trichoplusia ni (Lepidoptera: Noctuidae) that frequently feeds on glucosinolate-containing plants. Caterpillars feeding on diets with high concentrations of ITC experienced reduced survivorship and growth rates. High concentrations of ITC suppressed the appearance of several types of hemocytes and melanization activity, which are critical defenses against parasitic Hymenoptera and microbial pathogens. In terms of T. ni humoral immunity, only the antimicrobial peptide (AMP) genes lebocin and gallerimycin were significantly upregulated in caterpillars fed on diets containing high levels of ITC relative to caterpillars that were provided with ITC-free diet. Surprisingly, challenging caterpillars with a non-pathogenic strain of Escherichia coli resulted in the upregulation of the AMP gene cecropin. Feeding on high concentrations of plant toxins hindered caterpillar development, decreased cellular immunity, but conferred mixed effects on humoral immunity. Our findings provide novel insights into the effects of herbivore diet composition on insect performance demonstrating the role of specific plant defense toxins that shape herbivore immunity and trophic interactions.

Morphology of internal organs and hemolymph of the bird-eating spiders Pterinochilus Murinus RCF (Araneae: Theraphosidae)

The goal of the research was to study the morphological and histological properties of the internal organs and the hemolymph content of tarantula Pterinochilus murinus RCF (Araneae: Theraphosidae). For histological examination, 5 tarantulas of the Pterinochilus murinus RCF species, at the age of L6 (the sixth molt), were used. Morphological studies of tissues, organs, and hemocyte populations were carried out using light microscopy. Macroscopic photos of the heart, liver, lymphoid organs were presented with a description of their shape, structure and topography. The biomaterial was processed in accordance with the method of preparation of histological preparations, which included fixation, washing, dehydration, xylene and paraffin embeddings, preparation of histological sections, deparaffinization and staining with hematoxylin and eosin. Tissues and individual morphological cellular structures of the heart, liver, and lymphoid organs of tarantulas have been described. Also, smears of the spider hemolymph were made, which were fixed on a glass slide and stained by the Romanovsky-Giemsa method. The hemolymph of invertebrates is able to perform all the organism functions. During the study, several types of hemocytes were identified, which were classified primarily depending on morphological characteristics (cell size, shape, presence or absence of intracellular inclusions and their characteristics), the ability to be stained and the staining intensity of various morphological cell structures. The terminology of the described histological structures was given in accordance with the International Histological Nomenclature. Information about morphology of the organs, histological structure of the tissues and cells of tarantulas has been obtained expanding the knowledge of the Pterinochilus murinus RCF species.

Granules of immune cells are the source of organelles in the regenerated nerves of crayfish antennae.

Regeneration refers to the regrowing and replacing of injured or lost body parts. Crayfish antennae are nervous organs that are crucial for perceiving environmental signals. Immune cells (hemocytes) are responsible for neurogenesis in crayfish. Here, we used transmission electron microscopy to investigate at ultrastructural levels the potential roles of immune cells in nerve regeneration in crayfish antennae after amputation. The results showed that, while all three types of hemocytes were observed during nerve regeneration, granules of semi-granulocytes and granulocytes are the main sources of new organelles such as mitochondria, the Golgi apparatus and nerve fibres in the regenerated nerves of crayfish antennae. We describe the transformation of immune cell granules into different organelles in the regenerating nerve at ultrastructural levels. Also, we observed that the regeneration process speeds up after crayfish moulting. In conclusion, the granules are compacted packages of versatile materials carried by immune cells and can be converted into different organelles during nerve regeneration in crayfish antennae.

Single-Nucleus Sequencing of Fat Body Reveals Distinct Metabolic and Immune Response Landscapes in Silkworm Larvae after Bombyx mori Nucleopolyhedrovirus Infection.

The fat body plays a central role in the regulation of the life cycle of insects and acts as the major site for detoxification, nutrient storage, energy metabolism, and innate immunity. However, the diversity of cell types in the fat body, as well as how these cell subsets respond to virus infection, remains largely unknown. We used single-nucleus RNA sequencing to identify 23 distinct clusters representing adipocyte, hemocyte, epithelial cell, muscle cell, and glial cell types in the fat body of silkworm larvae. Further, by analysis of viral transcriptomes in each cell subset, we reveal that all fat body cells could be infected by Bombyx mori nucleopolyhedrovirus (BmNPV) at 72 h postinfection, and that the majority of infected cells carried at least a medium viral load, whereas most cells infected by BmNPV at 24 h postinfection had only low levels of infection. Finally, we characterize the responses occurring in the fat body cell clusters on BmNPV infection, which, on one hand, mainly reduce their metabolic functions, involving energy, carbohydrates, lipids, and amino acids, but, on the other hand, initiate a strong antiviral response. Our single-nucleus RNA sequencing analysis reveals the diversity of insect fat body cells and provides a resource of gene expression profiles for a systems-level understanding of their response to virus infection.

Morphological Analysis of the Hemolymph Cell Composition in the Bivalve Mollusk <i>Anadara</i> <i>broughtonii</i> Schrenck, 1867 (Sea of Japan)

The hemolymph cells of the ark clam Anadara broughtonii were examined using light microscopy, flow cytometry and gradient centrifugation. All three methods of analysis made it possible to identify two main types of cells in the hemolymph of the ark clam Anadara broughtonii – large granular erythrocytes and small agra-nular amebocytes. Erythrocytes accounted for 95.6 ± 0.9% of the total number of hemolymph cells. Erythrocytes were hemoglobin-containing cells with a great number of granules in the cytoplasm, a low nuclear-cytoplasmic ratio (NCR) and a lower intensity of cellular respiration compared to amebocytes. Amebocytes are cells of predominantly irregular shape with a high number of cells that do not contain or contain no more than 10 granular inclusions in the cytoplasm. All types of hemocytes found in the ark clam hemolymph demonstrated the same ability to spontaneously produce reactive oxygen species. For the first time, it has been shown that red blood cells of the ark clam Anadara broughtonii are capable of phagocytosis. At the same time, amebocytes absorbed on the average two times more zymosan particles (10.3 ± 0.7 pcs.), compared to erythrocytes (5.3 ± 0.1 pcs.).

MORPHOLOGICAL AND QUANTITATIVE STUDIES OF THE HEMOCYTES IN NYMPH AND IMAGO OF BLAPTICA DUBIA (SERVILLE, 1839)

The purpose of the research is to analyze how the hemocyte counts and their morphological characteristics change during development of Blaptica dubia, especially during molting. And to suggest a possible scheme of the transformation of spherulocytes into crescent-cells.
 Materials and methods. Quantitative and morphological features of hemocytes of imago and nymphs of 1-4 stages of Blaptica dubia using light microscopy and AFM were researched. Cell, nucleus and granule sizes were determined using NIS-Elements and NOVA software. Cytochemical tests with neutral red and alcyan blue allowed us to identify different cell types.
 Results. This study presents a classification of hemocytes in Blaptica dubia, that based on morphological and cytochemical characteristics of cells. We analyzed the dynamics of the hemocytes counts in 6 nymphs (1-4 stages) and 6 imago. Special attention is given to the moment of molting. Seven hemocyte types were described and identified as: prohemocytes, plasmocytes, granulocytes, vermicytes, spherulocytes, coagulocytes and crescent-cells. The stem cells (prohemocytes) and spherulocytes were the predominant hemocytes. Prohemocytes counts decrease as a result of insect development, instead the relative number of plasmocytes and granulocytes increases. Spherulocytes counts could increase to 35% at the moment of leaving the ootheca and at the molting period. Due to the study of hemocytes diversity in different nymph stages, interim cells and possible ways of their transformations were identified. The unusual crescent-cells, that were unique for Gromphadorhina portentosa, had been found in Blaptica dubia. For the first time, the cytochemical test allowed to determine the origin of crescent-cells from spherulocytes.

Antixenosis of Thodan (AAB), a resistant Musa cultivar to Odoiporus longicollis (Olivier) and characterization of larvicide molecules

Banana pseudostem borer (BPB) Odoiporus longicollis (Olivier) (Coleoptera, Curculionidae) is a serious pest of Musa cultivars. Experimental maintenance of larvae in the live pseudostem of cultivar Thodan, a resistant AAB Musa cultivar has resulted antixenosis followed by death of larvae within a week. Antixenosis was characterized by significant decrease of total hemocytes and sharp changes on the proportionate distribution of different types of hemocytes in larvae. As the number of plasmatocytes, prohemocytes, splenocytes and adipohemocytes decreased the number of granulocytes and oenocytes increased. Antixenosis also caused accumulation of 20-hydroxyecdysone (20E) and significant inhibition on the activities of trypsin like serine protease (TlSP) and phenoloxidase (PO). Phytochemical analysis of Thodan resulted characterization of three larvicides such as Betulinic acid (BA), Stigmasterol-3-O-glucoside (SOG) and Sulfoquinovosyl diacyl glycerol (SQDG), and the content in the pseudostem ranged 0.0027 to 0.007 per cent. All the three larvicides were highly toxic to the larvae with LD50 of 0.38 ppm for SOG, 0.41 ppm for SQDG and 0.83 ppm for BA. Simultaneous action of three larvicides in the live pseudostem resulted resistance in Thodan against infestation by BPB. Susceptible Nendran showed negligibly low content of SOG (0.0011%) and SQDG (0.0013%) but no detectable quantity of BA. Intoxication by all the three larvicides caused significant changes on the proportionate distribution of hemocytes, accumulation of 20-hydroxy ecdysone (20E) and inhibition of TlSP and PO, the enzymes involved in larval metamorphosis and cuticle sclerotisation. This study demonstrated that resistance of Thodan against O. longicollis is due to adverse effect of these larvicides on endocrine system, cuticle development and cytotoxicity of hemocytes. As these larvicide molecules are stable compounds, there is scope for them to be used as substitutes in place of deleterious insecticides for the management BPB.