Insect Hemocytes Research Articles

Characterization of integrins in cellular immunity of the oriental armyworm, Mythimna separata.

Insect hemocytes eliminate foreign substances from the hemocoel through various immune reactions. Integrins, receptor proteins present on the cell membrane, are formed as a heterodimer from α and β subunits and are known to be involved in various immune reactions. To elucidate the role of integrins in the immunity of the lepidoptera Mythimna separata, genes encoding integrins were screened from the genome, resulting in the identification of eight α and four β integrin genes. The expression levels of the integrin genes did not change in response to the injection of small abiotic beads undergoing phagocytosis in M. separata larvae. However, significant inductions of some integrin gene expressions were observed in hemocytes that formed capsules around large abiotic beads during encapsulation, especially in MysIntα2. Under biotic stimulation, induction of the MysIntα2 was evident after exposures to Gram-negative bacteria (Escherichia coli) and entomopathogenic nematodes (Steinernema carpocapsae), but not to Gram-positive bacteria (Micrococcus luteus). Immunostaining analysis revealed that MysIntα2 was specifically localized to hemocytes surrounding the beads during the encapsulation reaction. Furthermore, the spreading and encapsulation abilities of hemocytes were significantly inhibited by incubation with MysIntα2 antibodies. Suppression of MysIntα2 expression in M. separata larvae by injecting double-stranded RNA also resulted in a decrease in encapsulation activity. Collectively, these results indicate that MysIntα2 plays pivotal roles in the cellular immune response of M. separata, particularly during encapsulation. This likely occurs through the regulation of hemocyte spreading activity, thereby facilitating the formation of multilayered capsules around large invaders.

The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae.

In response to the replace mammal research models with insects in preliminary immunological studies, interest has grown in invertebrate defense systems. The immunological response is regulated by cytokines; however, while their role in mammals is well understood, little is known of their function in insects. A suitable target for studies into insect immunology is Galleria mellonella (Lepidoptera), the wax moth: a common host for human fungal and bacterial pathogens. G. mellonella is also a perfect subject for studies into the presence of cytokine-like proteins. The main goal of present research was detection in insect immunocompetent cells the 18 mammalian cytokines (IL-1α, IL-1β, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-β, GM-CSF, M-CSF, G-CSF), which play important role in immunological response and indication how their level change after fungal infection. The changes of cytokine-like proteins level were detected in hemocytes taken from G. mellonella larvae infected with entomopathogenic fungus, C. coronatus. The presence of cytokine-proteins was confirmed with using fluorescence microscopy (in cultured hemocytes) and flow cytometry (in freshly collected hemolymph). The ELISA test was used to detect changes in concentration of examined cytokine-like proteins. Our findings indicated the presence of eighteen cytokine-like molecules in G. mellonella hemocytes during infection with C. coronatus. The hemocytes taken from infected larvae demonstrated higher fluorescence intensity for six cytokine-like proteins (GM-CSF, M-CSF, IL-3, IL-15, IL-1β and IL-19) compared to untreated controls. ELISA test indicated significantly higher IL-3 and IL-15. M-CSF, IL-1α and IL-19 concentration in the hemolymph after fungal infection, and significantly lower TNF-β and G-CSF. Our findings confirm that the selected cytokine-like molecules are present in insect hemocytes and that their concentrations change after fungal infection, which might suggest that they play a role in the anti-fungal immunological response.

SfMBP: A novel microbial binding protein and pattern recognition receptor in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

The fall armyworm, Spodoptera frugiperda, poses a significant threat as a highly destructive agricultural pest in many countries. Understanding the complex interplay between the insect immune system and entomopathogens is critical for optimizing biopesticide efficacy. In this study, we identified a novel microbial binding protein, SfMBP, in S. frugiperda. However, the specific role of SfMBP in the immune response of S. frugiperda remains elusive. Encoded by the LOC118269163 gene, SfMBP shows significant induction in S. frugiperda larvae infected with the entomopathogen Beauveria bassiana. Consisting of 115 amino acids with a signal peptide, an N-terminal flexible region and a C-terminal β-sheet, SfMBP lacks any known functional domains. It is expressed predominantly during early larval stages and in the larval epidermis. Notably, SfMBP is significantly induced in larvae infected with bacteria and fungi and in SF9 cells stimulated by peptidoglycan. While recombinant SfMBP (rSfMBP) does not inhibit bacterial growth, it demonstrates binding capabilities to bacteria, fungal spores, peptidoglycan, lipopolysaccharides, and polysaccharides. This binding is inhibited by monosaccharides and EDTA. Molecular docking reveals potential Zn2+-interacting residues and three cavities. Furthermore, rSfMBP induces bacterial agglutination in the presence of Zn2+. It also binds to insect hemocytes and SF9 cells, enhancing phagocytosis and agglutination responses. Injection of rSfMBP increased the survival of S. frugiperda larvae infected with B. bassiana, whereas blocking SfMBP with the antibody decreased survival. These results suggest that SfMBP acts as a pattern recognition receptor that enhances pathogen recognition and cellular immune responses. Consequently, this study provides valuable insights for the development of pest control measures.

Changes in histamine, HSF1, Cysteinyl leukotriene, TLR1 and TLR2 in Galleria mellonella hemolymph after Conidiobolus coronatus infection

Non-mammal models are gaining popularity due to their ease of maintenance, low cost and lack of ethical restrictions. As some processes taking place in insect hemocytes are mirrored in human immunocytes, many immunological studies are now turning to the use of insect models like Galleria mellonella. G. mellonella larvae are susceptible to infection by Conidiobolus coronatus: an entomopathogenic fungus causing rhinofacial mycosis in humans. The study examines G. mellonella larvae during C. coronatus infection for the presence of certain compounds known to be involved in the human immunological response (histamine, HSF1, Cysteinyl leukotriene TLR1 and TLR2). G. mellonella larvae were exposed to fully grown fungus, and the results were determined by ELISA tests, fluorescence microscopy and flow cytometry. CysLT showed no significant changes between the control group and the infected larvae. Both histamine and HSF1 levels increased significantly after exposure to C. coronatus. Immunolocalization and flow cytometry of TLR1 and TLR2 proves that both receptors increase their levels due to the fungus infection. Our findings show that the cellular response of the G. mellonella is similar to the human response, indicating that G. mellonella larvae may be considered as a human-like immunological model in further studies.

The Changes in Mitochondrial Morphology and Physiology Accompanying Apoptosis in Galleria mellonella (Lepidoptera) Immunocompetent Cells during Conidiobolus coronatus (Entomophthorales) Infection.

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, their role in insects is not fully understood; thus, more indepth studies of insect cell apoptosis are necessary. The present study investigates mitochondrial involvement during Conidiobolus coronatus-induced apoptosis in Galleria mellonella hemocytes. Previous research has shown that fungal infection could induce apoptosis in insect hemocytes. Our findings indicate that mitochondria undergo several morphological and physiological changes during fungal infection, e.g., loss of mitochondrial membrane potential, megachannel formation, disturbances in intracellular respiration, increased nonrespiratory oxygen consumption in mitochondria, decreased ATP-coupled oxygen consumption and increased non-ATP-coupled oxygen consumption, decreased extracellular and intracellular oxygen consumption, and increased extracellular pH. Our findings confirm that G. mellonella immunocompetent cells demonstrate Ca2+ overload in mitochondria, translocation of cytochrome c-like protein from mitochondrial to cytosol fraction, and higher activation of caspase-9-like protein after C. coronatus infection. Most importantly, several of the changes observed in insect mitochondria are similar to those accompanying apoptosis in mammalian cells, suggesting that the process is evolutionarily conserved.

Beyond Cellular Immunity: On the Biological Significance of Insect Hemocytes.

Insect immunity is assorted into humoral and cellular immune reactions. Humoral reactions involve the regulated production of anti-microbial peptides, which directly kill microbial invaders at the membrane and intracellular levels. In cellular immune reactions, millions of hemocytes are mobilized to sites of infection and replaced by hematopoiesis at a high biological cost after the immune defense. Here, we considered that the high biological costs of maintaining and replacing hemocytes would be a better investment if hemocytes carried out meaningful biological actions unrelated to cellular immunity. This idea allows us to treat a set of 10 hemocyte actions that are not directly involved in immunity, some of which, so far, are known only in Drosophila melanogaster. These include (1) their actions in molting and development, (2) in surviving severe hypoxia, (3) producing phenoloxidase precursor and its actions beyond immunity, (4) producing vitellogenin in a leafhopper, (5) recognition and responses to cancer in Drosophila, (6) non-immune actions in Drosophila, (7) clearing apoptotic cells during development of the central nervous system, (8) developing hematopoietic niches in Drosophila, (9) synthesis and transport of a lipoprotein, and (10) hemocyte roles in iron transport. We propose that the biological significance of hemocytes extends considerably beyond immunity.

Inhibition of Dopamine Activity and Response of Rhipicephalus microplus Challenged with Metarhizium anisopliae.

Dopamine modulates ticks and insect hemocytes and links these arthropods' nervous and immune systems. For the first time, the present study analyzed the effect of a dopamine receptor antagonist on the survival, biological parameters, phagocytic index, and dopamine detection in the hemocytes of ticks challenged by Metarhizium anisopliae. The survival and egg production index of Rhipicephalus microplus were negatively impacted when ticks were inoculated with the antagonist and fungus. Five days after the treatment, the survival of ticks treated only with fungus was 2.2 times higher than ticks treated with the antagonist (highest concentration) and fungus. A reduction in the phagocytic index of hemocytes of 68.4% was observed in the group inoculated with the highest concentration of the antagonist and fungus compared to ticks treated only with fungus. No changes were detected in the R. microplus levels of intrahemocytic dopamine or hemocytic quantification. Our results support the hypothesis that dopamine is crucial for tick immune defense, changing the phagocytic capacity of hemocytes and the susceptibility of ticks to entomopathogenic fungi.

The immunotoxicity of ten insecticides against insect hemocyte cells in vitro.

Hemocytes in the hemolymph ofinsectsperform innateimmunity, but systematic studies to compare immunotoxicity of pesticides on hemocytes are still few. In this study, an insect hemocyte system was used to assess the impact of pesticides with different modes of action, which included loss of cell viability, inhibition of hemophagocytosis, and reduction of nitricoxidesynthase (NOS) activity. Results showed that piericidin A was the most cytotoxic to hemocytes, chlorfluazuron and hexaflumuron were the next. Also, piericidin A, chlorfenapyr, and fipronil had strong inhibitory effects on hemophagocytosis, and the effects of piericidin A and chlorfenapyr were persistent, while that of fipronil was short-lived. Moreover, fenoxycarb and hexaflumuron selectively inhibited granulocyte phagocytosis, tebufenozide only showed inhibition on plasmatocyte phagocytosis, but both inhibitory effects were transient. Furthermore, fenoxycarb and hexaflumuron showed a short-term strong inhibitory effect on the activity of NOS, chlorfenapyr and piericidin A showed a weak induction of NOS activity, while other pesticides exhibited a strong induction. Taken together, piericidin A was the most toxic and imidacloprid was the least toxic to hemocytes, and the alterations in hemocyte functions compromised immunity.

Intracellular cytokine detection based on flow cytometry in hemocytes from Galleria mellonella larvae: A new protocol.

Invertebrates are becoming increasingly popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. However, the use of insects as research models requires the development of methods for working with hemocytes. The aim of this study was to develop a protocol for intracellular cytokine detection in Galleria mellonella larvae hemocytes based on flow cytometry. It describes the anticoagulant composition of the buffer, the optimal conditions for hemocyte permeabilization and fixation, as well as the conditions of cell centrifugation to prevent cell disintegration. A key element is the selection of staining conditions, especially the length of the incubation time with the primary antibody, which turned out to be much longer than recommended for mammalian cells. The development of these individual steps allowed for the creation of a reproducible protocol for cytokine detection using flow cytometry in wax moth hemocytes. This will certainly facilitate the development of further protocols allowing for wider use of insect cells in immunological research.

The Role of Mosquito Hemocytes in Viral Infections.

Insect hemocytes are the only immune cells that can mount a humoral and cellular immune response. Despite the critical involvement of hemocytes in immune responses against bacteria, fungi, and parasites in mosquitoes, our understanding of their antiviral potential is still limited. It has been shown that hemocytes express humoral factors such as TEP1, PPO, and certain antimicrobial peptides that are known to restrict viral infections. Insect hemocytes also harbor the major immune pathways, such as JAK/STAT, TOLL, IMD, and RNAi, which are critical for the control of viral infection. Recent research has indicated a role for hemocytes in the regulation of viral infection through RNA interference and autophagy; however, the specific mechanism by which this regulation occurs remains uncharacterized. Conversely, some studies have suggested that hemocytes act as agonists of arboviral infection because they lack basal lamina and circulate throughout the whole mosquito, likely facilitating viral dissemination to other tissues such as salivary glands. In addition, hemocytes produce arbovirus agonist factors such as lectins, which enhance viral infection. Here, we summarize our current understanding of hemocytes’ involvement in viral infections.

Integrin β2 and β3: Two plasmatocyte markers deepen our understanding of the development of plasmatocytes in the silkworm Bombyx mori.

Insect hemocytes play important biological roles at developmental stages, metamorphosis, and innate immunity. As one of the most abundant cell types, plasmatocytes can participate in various innate immune responses, especially in encapsulation and node formation. Here, 2 molecular markers of plasmatocytes, consisting of integrin β2 and β3, were identified and used to understand the development of plasmatocytes. Plasmatocytes are widely distributed in the hematopoietic system, including circulating hemolymph and hematopoietic organs (HPOs). HPOs constantly release plasmatocytes with high proliferative activity in vitro; removal of HPOs leads to a dramatic reduction in the circulating plasmatocytes, and the remaining plasmatocytes gradually lose their ability to proliferate in vivo. Our results demonstrated that the release of plasmatocytes from HPOs is regulated by insulin-mediated signals and their downstream pathways, including PI3K/Akt and MAPK/Erk signals. The insulin/PI3K/Akt signaling pathway can significantly irritate the hematopoiesis, and its inhibitor LY294002 could inhibit the hemocytes discharged from HPOs. While the insulin/MAPK/Erk signaling pathway plays a negative regulatory role, inhibiting its activity with U0126 can markedly promote the discharge of plasmatocytes from HPOs. Our results indicate that the circulating plasmatocytes are mainly generated and discharged by HPOs. This process is co-regulated by the PI3K/Akt and MAPK/Erk signals in an antagonistic manner to adjust the dynamic balance of the hemocytes. These findings can enhance our understanding of insect hematopoiesis.

Hemocyte Clusters Defined by scRNA-Seq in Bombyx mori: In Silico Analysis of Predicted Marker Genes and Implications for Potential Functional Roles.

Within the hemolymph, insect hemocytes constitute a heterogeneous population of macrophage-like cells that play important roles in innate immunity, homeostasis and development. Classification of hemocytes in different subtypes by size, morphology and biochemical or immunological markers has been difficult and only in Drosophila extensive genetic analysis allowed the construction of a coherent picture of hemocyte differentiation from pro-hemocytes to granulocytes, crystal cells and plasmatocytes. However, the advent of high-throughput single cell technologies, such as single cell RNA sequencing (scRNA-seq), is bound to have a high impact on the study of hemocytes subtypes and their phenotypes in other insects for which a sophisticated genetic toolbox is not available. Instead of averaging gene expression across all cells as occurs in bulk-RNA-seq, scRNA-seq allows high-throughput and specific visualization of the differentiation status of individual cells. With scRNA-seq, interesting cell types can be identified in heterogeneous populations and direct analysis of rare cell types is possible. Next to its ability to profile the transcriptomes of individual cells in tissue samples, scRNA-seq can be used to propose marker genes that are characteristic of different hemocyte subtypes and predict their functions. In this perspective, the identities of the different marker genes that were identified by scRNA-seq analysis to define 13 distinct cell clusters of hemocytes in larvae of the silkworm, Bombyx mori, are discussed in detail. The analysis confirms the broad division of hemocytes in granulocytes, plasmatocytes, oenocytoids and perhaps spherulocytes but also reveals considerable complexity at the molecular level and highly specialized functions. In addition, predicted hemocyte marker genes in Bombyx generally show only limited convergence with the genes that are considered characteristic for hemocyte subtypes in Drosophila.

Ontogenetic features of the morphology of hemolymph cells in Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) as an indicator of biodiversity

Insect hemocytes are a complex of different types of cells that differ morphologically and functionally. The study of morphological variability of hemocytes Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) was carried out, eight types of cells were identified: spherulocytes, enocytoids, adipohemocytes, prohemocytes, plasma cells, granulocytes, cystocytes and phagocytes. The predominant type of hemocytes at all stages of ontogenesis are prohemocytes. In the hemolymph of larvae, the most numerous are prohemocytes with a basophilic nucleus (32.07%), prohemocytes with an eosinophilic nucleus (38.12%), and plasmatocytes (19.4%). The pupa is characterized by prohemocytes (64.3%), the sizes of which coincided with those of the adults (10.09 ± 2.11 and 9.45 ± 1.85 µm). At the adult stage, adipohemocytes, enocytoids and cystocytes were found, which were not observed at the larval stage. The size of hemocytes changed depending on the stage of ontogenesis. Thus, it was found that the cellular composition of the Henosepilachna vigintioctomaculata hemolymph is the same for the adult and pupal stages, but it has differences in the dimensional characteristics of the formed elements. The larval stage has its own characteristics in the cellular composition of the hemolymph: the absence of three types of hemocytes and the formation of two subtypes of prohemocytes.

MORPHOFUNCTIONAL ANALYSIS OF HEMOCYTES IN INSECTS (ORTHOPTERA) COLLECTED FROM MURTI AND ADJOINING AREAS (DOOARS) AND DIFFERENT AGRICULTURAL FIELDS OF NORTH 24 PARGANAS, WEST BENGAL

A small village named Murti is situated along the bank of river Murti in Dooars. The adjacent area of murti is rich in insect biodiversity. Knowledge on the diversity and the role of Orthopteran insects in tea ecosystem of West Bengal including Dooars and Terai is very scanty. Hemocytes are the chief immune cells of insects. The number and shape of hemocytes can change rapidly in response to environmental stress and pollution. Grasshopper (Orthoptera) species were collected from Murti and adjoining areas and from different agricultural fields of North 24 Parganas (West Bengal). Hemolymph was stained by Giemsa, and Neutral Red. Cellular morphology and phagocytosis was examined. Normal cell shape and behavioural activities like phagocytosis, cell-cell fusion was observed in insects collected from Murti, and adjoining areas of Dooars. But in case of insect hemocytes isolated from different agricultural fields of North 24 Parganas and its adjacent sites showed altered shape and different phases of cell death like degeneration of nuclei, formation of membrane blebs and rupture of plasma membrane. Toxic environmental pollutants may be responsible for alteration of hemocytes size and function which may effect on biodiversity. Hemocytes can be used as an effective bio-indicator by which the health of the ecosystem can be screened.

How Dopamine Influences Survival and Cellular Immune Response of Rhipicephalus microplus Inoculated with Metarhizium anisopliae

Dopamine (DA) is a biogenic monoamine reported to modulate insect hemocytes. Although the immune functions of DA are known in insects, there is a lack of knowledge of DA’s role in the immune system of ticks. The use of Metarhizium anisopliae has been considered for tick control, driving studies on the immune response of these arthropods challenged with fungi. The present study evaluated the effect of DA on the cellular immune response and survival of Rhipicephalus microplus inoculated with M. anisopliae blastospores. Exogenous DA increased both ticks’ survival 72 h after M. anisopliae inoculation and the number of circulating hemocytes compared to the control group, 24 h after the treatment. The phagocytic index of tick hemocytes challenged with M. anisopliae did not change upon injection of exogenous DA. Phenoloxidase activity in the hemolymph of ticks injected with DA and the fungus or exclusively with DA was higher than in untreated ticks or ticks inoculated with the fungus alone, 72 h after treatment. DA was detected in the hemocytes of fungus-treated and untreated ticks. Unveiling the cellular immune response in ticks challenged with entomopathogenic fungi is important to improve strategies for the biological control of these ectoparasites.

Galleria mellonella (Lepidoptera: Pyralidae) Hemocytes Release Extracellular Traps That Confer Protection Against Bacterial Infection in the Hemocoel.

Extracellular traps (ETs) released from vertebrate and invertebrate immune cells consist of chromatin and toxic granule contents that are capable of immobilizing and killing microbes. This recently described innate immune response is not well documented in insects. The present study found that ETs were released by hemocytes of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) in vivo and ex vivo after bacterial stimulation. ET release (ETosis), hemolymph coagulation, and melanization likely contributed to the immobilization and killing of the bacteria. The injection of G. mellonella hemocyte deoxyribonucleic acid (DNA) in the presence of bacteria increased bacterial clearance rate and prolonged insect survival. Taken together, these results indicate the presence of insect hemocyte extracellular traps (IHETs) that protect the insect against microbial infection in the hemocoel and represent the first documentation of ETs in insects in vivo.

Haemocyte variations in 35 species of grasshoppers and locusts.

Grasshoppers and locusts are widely distributed worldwide, causing significant losses in agriculture. The origin and functions of their haemocytes are not entirely understood. Insect haemocytes arbitrate cellular defence and participate in humoral defences. Due to their importance, the haemocytes of 35 species of grasshoppers and locusts from China were morphologically examined in this study. We aim to highlight a simple method for the morphological examination of insect haemocytes. The haemocytes were observed, counted and compared under a light microscope after Wright-Giemsa staining. High complexity in form and shape were observed in the haemocytes. These include prohaemocytes, plasmatocytes, granulocytes, vermicytes, podocytes and megakaryocytes. No clear relationship was seen between the haemocyte type and their phylogenetic relationship among the three families examined. The high abundance of plasmatocytes and granulocytes suggests their importance in the immunity of grasshoppers and locusts. The minor haemocyte populations including prohaemocytes, vermicytes and podocytes may not be always present in individuals. All examined species shared similarities in their haemocyte types. Wright-Giemsa staining is a simple and efficient method for evaluating haemocytes.

Drosophila as a Model to Study Cellular Communication Between the Hematopoietic Niche and Blood Progenitors Under Homeostatic Conditions and in Response to an Immune Stress.

In adult mammals, blood cells are formed from hematopoietic stem progenitor cells, which are controlled by a complex cellular microenvironment called “niche”. Drosophila melanogaster is a powerful model organism to decipher the mechanisms controlling hematopoiesis, due both to its limited number of blood cell lineages and to the conservation of genes and signaling pathways throughout bilaterian evolution. Insect blood cells or hemocytes are similar to the mammalian myeloid lineage that ensures innate immunity functions. Like in vertebrates, two waves of hematopoiesis occur in Drosophila. The first wave takes place during embryogenesis. The second wave occurs at larval stages, where two distinct hematopoietic sites are identified: subcuticular hematopoietic pockets and a specialized hematopoietic organ called the lymph gland. In both sites, hematopoiesis is regulated by distinct niches. In hematopoietic pockets, sensory neurons of the peripheral nervous system provide a microenvironment that promotes embryonic hemocyte expansion and differentiation. In the lymph gland blood cells are produced from hematopoietic progenitors. A small cluster of cells called Posterior Signaling Centre (PSC) and the vascular system, along which the lymph gland develops, act collectively as a niche, under homeostatic conditions, to control the balance between maintenance and differentiation of lymph gland progenitors. In response to an immune stress such as wasp parasitism, lymph gland hematopoiesis is drastically modified and shifts towards emergency hematopoiesis, leading to increased progenitor proliferation and their differentiation into lamellocyte, a specific blood cell type which will neutralize the parasite. The PSC is essential to control this emergency response. In this review, we summarize Drosophila cellular and molecular mechanisms involved in the communication between the niche and hematopoietic progenitors, both under homeostatic and stress conditions. Finally, we discuss similarities between mechanisms by which niches regulate hematopoietic stem/progenitor cells in Drosophila and mammals.

Effects of Copper Oxide Nanoparticles on Immune and Metabolic Parameters of Galleria mellonella L.

In this study, the effects of dietary CuO nanoparticles (NPs) on metabolic enzyme activity, biochemical parameters, and total (THC) and differential hemocyte counts (DHC) were determined in Galleria mellonella larvae. Using concentrations of 10, 100, 1000mg/L and the LC10 and LC30 levels of CuO NPs, we determined that the NPs negatively impacted metabolic enzyme activity and biochemical parameters in larval hemolymph. Compared with the control, the greatest increase in THC was observed in larvae fed on diets with 100mg L-1 of CuO NPs. Plasmatocytes and granulocytes were among the most numerous hemocytes in all treatments. These results suggest that dietary CuO NPs effects the metabolic metabolism and immune system of G. mellonella and provide indirect information regarding the toxic effects of CuO NPs in mammalian immune system given similarities between mammalian blood cells and insect hemocytes.

Eicosanoid‐induced calcium signaling mediates cellular immune responses of Tenebrio molitor

AbstractCalcium signaling is associated with actin cytoskeleton rearrangement. It is required for expressing cellular immune responses of insect hemocytes. Eicosanoids mediate immune responses by cytoskeleton rearrangement against various pathogen infections in insects. The objective of this study was to investigate cellular immune responses of a mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae). The hypothesis tested was that immune responses would be mediated by calcium signaling associated with eicosanoid mediation. Larvae of T. molitor possessed at least four morphological types of hemocytes, most (almost 75%) of which were granulocytes and plasmatocytes. They all exhibited a hemocyte‐spreading behavior by F‐actin extension. Upon bacterial infection, hemocytes exhibited phagocytosis and formed hemocytic nodules. Intracellular calcium was detected in hemocytes. Its amount increased with increasing incubation time after bacterial infection. Inhibition of calcium influx or release from endoplasmic reticulum using specific inhibitors prevented cellular immune responses. Interestingly, treatment with an eicosanoid biosynthesis inhibitor suppressed calcium signals induced by bacterial challenge. Furthermore, treatment with naproxen, a prostaglandin (PG) synthesis inhibitor, or esculetin, a leukotriene synthesis inhibitor, also inhibited the increase of calcium concentration in hemocytes. Among eicosanoids, PGE2 highly stimulated the increase of calcium signals. These results suggest that cellular immune responses of T. molitor are triggered by calcium signaling, which is up‐regulated by eicosanoids.