Number Of Hemocytes Research Articles

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster.

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling.

Immunosuppression in Honeybee Queens by the Neonicotinoids Thiacloprid and Clothianidin

Queen health is crucial to colony survival of honeybees, since reproduction and colony growth rely solely on the queen. Queen failure is considered a relevant cause of colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides can severely affect the immunocompetence of queens of western honeybees (Apis mellifera L.). In young queens exposed to thiacloprid (200 µg/l or 2000 µg/l) or clothianidin (10 µg/l or 50 µg/l), the total hemocyte number and the proportion of active, differentiated hemocytes was significantly reduced. Moreover, functional aspects of the immune defence namely the wound healing/melanisation response, as well as the antimicrobial activity of the hemolymph were impaired. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of queens, possibly leading to an impaired disease resistance capacity.

Insect Immunity Varies Idiosyncratically During Overwintering.

Overwintering insects face multiple stressors, including pathogen and parasite pressures that shift with seasons. However, we know little of how the insect immune system fluctuates with season, particularly in the overwintering period. To understand how immune activity changes across autumn, winter, and spring, we tracked immune activity of three temperate insects that overwinter as larvae: a weevil (Curculio sp., Coleoptera), gallfly (Eurosta solidaginis, Diptera), and larvae of the lepidopteran Pyrrharctia isabella. We measured baseline circulating hemocyte numbers, phenoloxidase activity, and humoral antimicrobial activity, as well as survival of fungal infection and melanization response at 12°C and 25°C to capture any potential plasticity in thermal performance. In Curculio sp. and E. solidaginis, hemocyte concentrations remained unchanged across seasons and antimicrobial activity against Gram-positive bacteria was lowest in autumn; however, Curculio sp. were less likely to survive fungal infection in autumn, whereas E. solidaginis were less likely to survive infection during the winter. Furthermore, hemocyte concentrations and antimicrobial activity decreased in P. isabella overwintering beneath snow cover. Overall, seasonal changes in activity were largely species dependent, thus it may be difficult to create generalizable predictions about the effects of a changing climate on seasonal immune activity in insects. However, we suggest that the relationship between the response to multiple stressors (e.g., cold and pathogens) drives changes in immune activity, and that understanding the physiology underlying these relationships will inform our predictions of the effects of environmental change on insect overwintering success.

Effect of Parasitization by Microplitis pallidipes Szepligeti on the Hemolymph Melanization, Total Number and Composition of Hemocytes in the Hemolymph of Larval Spodoptera exigua (Hübner)

Effect of Parasitization by Microplitis pallidipes Szepligeti on the Hemolymph Melanization, Total Number and Composition of Hemocytes in the Hemolymph of Larval Spodoptera exigua (Hübner)

Lactobacillus paracasei modulates the immune system of Galleria mellonella and protects against Candida albicans infection.

Probiotics have been described as a potential strategy to control opportunistic infections due to their ability to stimulate the immune system. Using the non-vertebrate model host Galleria mellonella, we evaluated whether clinical isolates of Lactobacillus spp. are able to provide protection against Candida albicans infection. Among different strains of Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus fermentum, we verified that L. paracasei 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans. We found that the injection of 107 cells/larvae of L. paracasei into G. mellonella larvae infected by C. albicans increased the survival of these insects compared to the control group (P = 0.0001). After that, we investigated the immune mechanisms involved in the protection against C. albicans infection, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that L. paracasei increased the hemocyte quantity (2.38 x 106 cells/mL) in relation to the control group (1.29 x 106 cells/mL), indicating that this strain is capable of raising the number of circulating hemocytes into the G. mellonella hemolymph. Further, we found that L. paracasei 28.4 upregulated genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, L. paracasei 28.4 increased gene expression of galiomicin by 6.67-fold and 17.29-fold for gallerymicin. Finally, we verified that the prophylactic provision of probiotic led to a significant reduction of the number of fungal cells in G. mellonella hemolymph. In conclusion, L. paracasei 28.4 can modulate the immune system of G. mellonella and protect against candidiasis.

Effect of inulin and fulvic acid on survival, growth, immune system, and WSSV prevalence in Litopenaeus vannamei

The effect of inulin and fulvic acid, added in the feed, on growth, survival, WSSV prevalence, and immune system was studied in Litopenaeus vannamei. To the above, a bioassay, with treatments in triplicate, was performed to test different additive concentrations. WSSV analysis was done in organisms infected with a relatively high viral load using single and nested PCR. At the end of the bioassay, hemolymph was extracted and the immune system was studied in hemocytes at biochemical and genetic level (quantitative PCR). The final growth was similar in all treatments and survival was between 66,7% and 93,3%. WSSV prevalence decreased 13% as compared to control. The number of hemocytes, phenoloxidase activity, and superoxide anion concentration were similar in all treatments. Inulin and fulvic acid modulated the expression of transglutaminase, superoxide dismutase, and prophenoloxidase genes, but not the Toll receptor. Additives do not negatively affect growth of white shrimp and they protect them against WSSV when infected with a relatively high viral load. Additives did not affect the immune system effectors studied at biochemical level but they modulated the expression of some immune-related genes in L. vannamei.

Effects of pyrene exposure on immune response and oxidative stress in the pearl oyster, Pinctada martensii

Pyrene is a polycyclic aromatic hydrocarbon (PAH) commonly observed in aquatic ecosystems, which originates primarily from the incomplete combustion of fossil fuels and the use of petroleum compounds. Pyrene can cause the immune disturbance and oxidative stress, result in immunotoxicity, DNA damage, reduce reproduction significantly, and induce behavioral changes. Marine bivalves are commonly used as bioindicators for marine pollution, and hemolymph is a metabolite transfer medium for PAH pollutant. However, the vital immune indicator responses of pearl oyster Pinctada martensii hemolymph exposed to pyrene is still unclear. Thus, the immunotoxic responses of pyrene on the hemolymph of the Pinctada martensii were investigated in this study. After exposure to pyrene for 7 days, the total number of hemocytes (THC), cell membrane stability (CMS), phagocytic activity (PA) and total glutathione (GSHT) all decreased significantly. Pyrene also caused a significant increase in lipid peroxidation (LPO). Median effective concentrations (EC50) of pyrene on THC (4.5 μg L−1) and LPO (5.2 μg L−1) were lower than those for CMS (13.8 μg L−1), PA (12.1 μg L−1) and GSHT (7.2 μg L−1), which indicates that THC and LPO were more sensitive. Additionally, a clear dose-effect relationship indicated that pyrene stimulated a marked immune response, as well as oxidative stress in P. martensii, which demonstrates the subtle effects of pyrene exposure on marine invertebrates and the potential associated risk.

Significant improvement of shrimp growth performance by growth hormone-releasing peptide-6 immersion treatments

Growth hormone-releasing peptide-6 (GHRP-6) is one of the earliest developed synthetic peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand ghrelin. In vertebrates, ghrelin is a potent circulating orexigenic hormone with functional roles in controlling food intake, energy expenditure, adiposity, growth hormone secretion and immunity. Ghrelin has been studied mainly in vertebrates; thus, little is known about its role in invertebrates, including crustaceans. We first evaluated the effect of GHRP-6 injection over feed intake in shrimp and its effects on shrimp growth when the peptide was administrated by successive immersion baths. GHRP-6 increased feed intake, body weight and size, the number of rostral spines and gill branches, protein concentration and haemocyte number in treated shrimps. We also evaluated the peptide uptake and clearance in a pharmacokinetics, using [H3]GHRP-6 administered to postlarvae. Given a limited exposure and efficient clearance of the peptide-associated radioactivity from larvae, our findings suggested that GHRP-6-treated Litopenaeus vannamei can be consumed safely by humans after aquaculture applications. These results propose that GHRP-6 could be an additional tool to study growth physiology in crustaceans and also a promising candidate for development into a new biotechnology product for improving shrimp growth and quality.

Regulation by gut bacteria of immune response, Bacillus thuringiensis susceptibility and hemolin expression in Plodia interpunctella

Plodia interpunctella (Hübner) is an important stored grain insect pest worldwide, and the first lepidopteran with reported resistance to Bacillus thuringiensis (Bt) toxins. Since gut bacteria may affect Bt insecticidal activity, we evaluated whether P. interpunctella lacking gut bacteria had differences in immune responses and susceptibility to the Bt formulation, Bactospeine. In order to clear gut bacteria, third instar larvae were reared on artificial diet containing antibiotics, or were obtained from sterilized eggs and reared under sterile conditions, and larvae were fed diets with or without Bt. Mortality was significantly lower (p<0.05) in bacteria-free larvae treated with Bt, compared with Bt-treated larvae with unaffected gut bacteria. The number of hemocytes was lower in control and Bt-treated larvae, but was significantly higher (p<0.001) in larvae treated with antibiotics and Bt, and larvae from presterilized eggs and reared on sterile diet had the highest number of hemocytes. Phenoloxidase activity was significantly lower (p<0.05) in Bt-treated larvae from presterilized eggs reared on antibiotics for 24h or in larvae reared on antibiotic-treated diets prior to Bt introduction compared with those fed control diet. Hemolin gene expression was reduced in larvae fed Bt diets compared with control and was not detected in larvae treated with antibiotics. Larvae from sterilized eggs and fed sterile diet never reached the pupal stage. Therefore, the loss of gut bacteria in P. interpunctella larvae affected the host immune response and expression of the hemolin gene, and significantly reduced susceptibility to Bt.

Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses

Adult common octopus individuals were intramuscularly infected with Photobacterium damsela subsp. piscicida in order to investigate if this species is sensitive to this common and important fish pathogen. The fate of the bacterial antigens and the tissue responses of Octopus vulgaris were studied employing immunohistochemical techniques.Strong reaction at the site of injection was evident from day 2 post-infection that continued until day 14. Great numbers of hemocytes that were attracted at the site of infection were involved in phagocytosis of bacteria. Very early in the infection, a transition of cells to fibroblasts and an effort to isolate the infection was observed. During the course of the study, very large necrotic cells were seen at the site of infection, whereas during the later stages hemocytes with phagocytosed bacteria were observed in well-defined pockets inside the muscle tissue. None of the internal organs tested for the presence of the bacterium were positive with the exception of the digestive gland where antigen staining was observed which was not associated with hemocyte infiltration. The high doses of bacterial cells used in this experimental infection and the lack of disease signs from Octopus vulgaris suggest that, under normal conditions, octopus is resistant to Photobacterium damsela subsp. piscicida.

Cellular and humoral responses of Pieris brassicae to infection by Steinernema feltiae, its symbiont bacteria, and their metabolites

This study describes the mortality effects of the culture broth ofXenorhabdus bovieniiand its aqueous and organic extracts onPieris brassicaelarvae. All the treatments had insecticidal activities when injected into the haemocoel. The culture broth of bacteria and its aqueous extracts exhibited more insecticidal activity. The results showed the immune response ofP. brassicaeexposed toSteinernema feltiae, its symbiont bacteria,X. bovienii, and aqueous and organic extracts of bacteria. Both cellular and humoral responses were investigated. After infection of the larvae ofP. brassicaewithS. feltiae, the total number of haemocytes quickly increased. A reduction in the number of haemocytes was observed over post-injection time. In addition, plasmatocytes and granulocytes showed increased frequency and significant changes when compared to other haemocytes in responding to entomopathogenic nematode injection.Steinernema feltiaeincreased phenoloxidase activity but 3 h post-injection the concentration gradually decreased. However, a reduction in phenoloxidase activity was observed when the larvae were infected with bacteria or their aqueous and organic extracts. This study showed that both living and heat-killed bacteria have suppression effects on phenoloxidase activity. The lysozyme concentration increased inP. brassicaelarvae when they were exposed to living and heat-killedX. bovienii. No suppression effect of the bacteria was detected on lysozyme.

Immunological Responses of Sesamia cretica to Ferula ovina Essential Oil

The current research was performed aiming to investigate the effects of Ferula ovina essential oil on the fourth instar larval hemogram of Sesamia cretica. Four main sorts of circulating hemocytes, including prohemocytes, plasmatocytes, granulocytes (GRs), and oenocytoides, were identified in the fourth instar larvae. Treatment of the larvae with the concentration of 1000 ppm of the essential oil led to an enhancement of the total hemocyte and GR count followed by a dose-dependent decrease at the concentrations of 2500 and 7000 ppm. Plasmatocyte numbers declined in all the treatments with more significant effects at increased doses. The greatest numbers of GRs, plasmatocytes, and total hemocytes were found after 48 h of treatment. The highest phenol-oxidase activity was recorded 12 h after treatment at the concentration of 2500 ppm. The highest effect on nodule formation was exerted by the concentration of 7000 ppm 12 h after treatment. The results of the present study clearly indicated that the treatment of larvae by the essential oil of F. ovina decreased the numbers of total and differential hemocyte counts although phenol-oxidase activity and the number of nodules showed no decline in the treated larvae. These results demonstrated that Ferula ovina essential oil has a significant effect on the immune ability of the studied insect and can be useful and usable for future research to practical management of this pest.

Infection of silkworm larvae by the entomopathogenic fungus Metarhizium anisopliae

ABSTRACT: The isolate E9 of Metarhizium anisopliae was used in commercial hybrids of Bombyx mori larvae to evaluate its biological effect. Symptomatological analyses showed typical signs of fungal infection. Histopathology revealed the presence of large numbers of hemocytes in the hemocoel, and on the sixth dpi the bodies of the insects appeared to be colonised by the fungus. The isolate E9 is pathogenic to larvae B. mori and; therefore, death of the insects was caused by the colonization of fungus in the epidermal and mesodermal tissues.

Effect of stress on heat shock protein levels, immune response and survival to fungal infection of Mamestra brassicae larvae

Although the utilisation of fungal biological control agents to kill insect pests is desirable, it is known that the outcome of infection may be influenced by a number of criteria, including whether or not the target insect is stressed. In the current work, topical treatment of larvae of the lepidopteran pest, Mamestra brassicae, with conidia of Beauveria bassiana, followed by a heat stress (HS; 37°C for 1h) 48h later, resulted in a similar level of larval survival to that occurring for no heat stress (No-HS), fungus-treated larvae. By contrast, when the HS was applied 24h after fungal treatment, larval survival was significantly increased, indicating that the HS is protecting the larvae from B. bassiana. Similarly, exposure of larvae to a HS provided protection against Metarhizium brunneum (V275) at 48h (but not 24h) after fungal treatment.To elucidate the mechanism(s) that might contribute to HS-induced increases in larval survival against fungal infection, the effects of a HS on key cellular and humoral immune responses and on the level of selected heat shock proteins (HSP) were assessed. When larvae were kept under control (No HS) conditions, there was no significant difference in the haemocyte number per ml of haemolymph over a 24h period. However, exposure of larvae to a HS, significantly increased the haemocyte density immediately after (t=0h) and 4h after HS compared to the No HS controls, whilst it returned to control levels at t=24h. In addition, in vitro assays indicated that haemocytes harvested from larvae immediately after (0h) and 4h (but not 24h) after a HS exhibited higher rates of phagocytosis of FITC-labelled B. bassiana conidia compared to haemocytes harvested from non-HS larvae. Interestingly, the HS did not appear to increase anti-fungal activity in larval plasma. Western blot analysis using antibodies which cross react with Drosophila melanogaster HSP, resulted in a relatively strong signal for HSP 70 and HSP 90 from extracts of 50,000 and 100,000haemocytes, respectively, harvested from No-HS larvae. By contrast, for HSP 60, a lysate derived from 200,000haemocytes resulted in a relatively weak signal. When larvae were exposed to a HS, the level of all three HSP increased compared to the No HS control 4h and 16h after the HS. However, 24h after treatment, any heat stress-mediated increase in HSP levels was minimal and not consistently detected. Similar results were obtained when HSP 90, 70, and 60 levels were assessed in fat body harvested from heat stressed and non-heat stressed larvae. With regard to HSP 27, no signal was obtained even when a lysate from 200,000haemocytes or three times the amount of fat body were processed, suggesting that the anti-HSP 27 antibody utilised does not cross-react with the M. brassicae HSP. The results suggest that a HS-mediated increase in haemocyte density and phagocytic activity, together with an upregulation of HSP 90 and 70, may contribute to increasing the survival of M. brassicae larvae treated with B. bassiana and M. brunneum (V275).

Modulation of immune and antioxidant responses by azinphos-methyl in the freshwater mussel Diplodon chilensis challenged with Escherichia coli.

The aim of the present study was to characterize the immune response-total hemocyte number, cell type proportion, hemocyte viability, lysosomal membrane stability, phagocytic activity, cellular acid and alkaline phosphatase activity, and humoral bacteriolytic and phenoloxidase activity--in Diplodon chilensis exposed to 0.2 mg/L of azinphos-methyl (AZM), using Escherichia coli as immunological and pro-oxidant challenges. In addition, glutathione-S-transferase and lipid peroxidation thiobarbituric acid reactive substances were analyzed in gill tissue. Mussels from an unpolluted site were treated for 3 d as follows: 1) experimental control; 2) solvent effects control (acetone 0.01%); 3) bacterial challenge effects control (E. coli, 5 cells/mL × 104 cells/mL); 4) pesticide effects control (AZM in acetone); 5) control for combined effects of solvent and bacterial challenge; and 6) exposed to AZM, then challenged with E. coli. The results showed increased granulocyte proportion and phagocytic activity. Partial reversion of deleterious effects of E. coli on lysosomal membranes was observed in mussels exposed to AZM and then challenged with E. coli. Total hemocyte number and humoral bacteriolytic activity were increased only by E. coli challenge. Acid phosphatase activity was increased by both E. coli and AZM, whereas the stimulating effect of E. coli on alkaline phosphatase activity was negatively modulated by AZM. Azinphos-methyl inhibited phenoloxidase activity regardless of the E. coli challenge. Gill glutathione-S-transferase activity was increased by E. coli treatment either alone or pretreated with acetone or AZM and by AZM alone. Thiobarbituric acid reactive substance levels were reduced by AZM alone or combined with the E. coli challenge and by acetone followed by the E. coli challenge. Both acetone and AZM seem to be important modulators of immune and antioxidant responses in D. chilensis. Environ Toxicol Chem 2017;36:1785-1794. © 2016 SETAC.

Haemocoel injection of PirA1B1 to Galleria mellonella larvae leads to disruption of the haemocyte immune functions.

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey. In this study, we cloned the gene coding for a binary toxin PirA1B1 and purified the recombinant protein using affinity chromatography combined with desalination technology. Furthermore, the cytotoxicity of the recombinant protein against the haemocytes of Galleria mellonella larvae was investigated. We found that the protein had haemocoel insecticidal activity against G. mellonella with an LD50 of 131.5 ng/larva. Intrahaemocoelic injection of PirA1B1 into G. mellonella resulted in significant decreases in haemocyte number and phagocytic ability. In in vitro experiments, PirA1B1 inhibited the spreading behaviour of the haemocytes of G. mellonella larvae and even caused haemocyte degeneration. Fluorescence microscope analysis and visualization of haemocyte F-actin stained with phalloidin-FITC showed that the PirA1B1 toxin disrupted the organization of the haemocyte cytoskeleton. Our results demonstrated that the PirA1B1 toxin disarmed the insect cellular immune system.

Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus

Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PlHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PlHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PlHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PlHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PlHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PlHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.

The cellular death pattern of primary haemocytes isolated from the black tiger shrimp (Penaeus monodon)

A key to successfully generate the penaeid shrimp cell line is to find out how primary cells died. The most suitable period to culture Penaeus monodon haemocytes was in the first 48 h of culture because cells had normal morphology, high percent of viable cells (65.29 ± 5.43%), low percent of early (11.75 ± 1.30%) and late apoptotic cells (15.47 ± 11.71%) determined by Annexin V and TUNEL including constant IAP (0.06 ± 0.01–0.07 ± 0.01) and caspase-3 expression (0.30 ± 0.06–0.39 ± 0.10) by real-time PCR throughout the experiment. Moreover, adding 50 and 250 μM of the cell permeable pan caspase inhibitor Z-VAD–FMK produced some melanised cells since the 48th hour, while percent of viable cells was decreased since the 24th hour with no difference in percent of early and late apoptotic cells compared to control at each time point. No difference of IAP and caspase-3 expression level in both Z-VAD–FMK groups was found compared to control and vehicle groups at each time point, excluding caspase-3 in 250 μM Z-VAD–FMK at the 24th hour was higher than control and vehicle. Supplementing sodium fluoride (NaF) induced cell membrane damage and cellular shrinkage of primary haemocytes within 2 h. Even percent of viable cells was reduced down to zero and percent of late apoptotic cells was increased by 2 h of incubation in 25 and 50 mM NaF, IAP and caspase-3 in all NaF groups was not different from control. These results indicate that a number of primary haemocytes derived in this study die through the apoptotic process.

Reactive Oxygen Species Affect Transglutaminase Activity and Regulate Hematopoiesis in a Crustacean

Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.

Cellular immune response of the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Pyralidae), to infection by the entomopathogenic fungus, Beauveria bassiana

The term cellular immune response refers to haemocyte-mediated responses, including phagocytosis, nodulation, and encapsulation. In the present study, we identified five types of circulating haemocytes in larvae of the haemolymph of the Asian corn borer, Ostrinia furnacalis (Guenee), including granulocytes, oenocytoids, plasmatocytes, prohaemocytes, and spherulocytes. The relative number of total free haemocytes per larva decreased significantly 0.5, 24, and 36 h after the injection of Beauveria bassiana conidia. Upon conidia challenge, both phagocytosis and nodulation were observed in the collected haemolymph from O. furnacalis larvae. In addition, plasma was found to be necessary for both phagocytosis and nodulation. Therefore, we here confirm that phagocytosis and nodulation are involved in O. funacalis larvae during their fight against infection by B. bassiana, and further, that the cellular immune response of O. furnacalis helps eliminate the invading organisms despite the fact that not all the fungal conidia are killed.