Types Of Hemocytes Research Articles

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

AbstractHemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi‐granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte‐based immune responses as an endpoint in ecotoxicology.

Morphological and functional characterization of the oyster Crassostrea gasar circulating hemocytes: Cell types and phagocytosis activity

Hemocytes are the circulating cells of the hemolymph of oysters and are responsible for numerous physiological functions, including immune defense. The oyster Crassostrea gasar is a native species inhabiting mangrove habitat and is of great commercial interest, cultured throughout the Brazilian coast, mainly in the north and northeast. Despite its commercial importance, little is known about its immunological aspects and defense cells, the hemocytes. This work aimed to morphologically characterize hemocytes of the oyster C. gasar and to study one of the main cellular defense response, phagocytosis, using light microscopy and flow cytometry. The results showed the presence of six hemocyte populations in C. gasar hemolymph. These comprise of large and small granulocytes, large and small hyalinocytes, blast-like cells and a rare type classified as vesicular or serous hemocytes. Hyalinocytes were highly abundant and the most heterogeneous cell population, while small granulocytes, along with vesicular hemocytes were the less abundant population. Hemocytes of C. gasar oysters demonstrated capabilities to phagocytose three different types of particles tested: zymosan A, latex particles and Escherichia coli, indicating a broad defense capacity. The zymosan A were the most engulfed particles, followed by beads, mainly phagocytized by granulocytes, the most phagocytic cells, and finally E. coli, which were the least phagocytized. This study is the first characterization of C. gasar oyster hemocytes and will support future studies that aim to understand the participation of different hemocyte types in defense responses against pathogens and/or environmental changes.

Hallmarks of crustacean immune hemocytes at single-cell resolution.

In invertebrates, hemocytes are the key factors in innate immunity. However, the types of invertebrate immune hemocytes are unclassified due to the limitation of morphological classification. To determine the immune hemocytes of crustaceans, the heterogeneity of hemocytes of shrimp Marsupenaeus japonicus and crayfish Procambarus clarkii, two representative crustacean species, were characterized in this study. The results of single-cell RNA sequencing indicated that shrimp and crayfish contained 11 and 12 types of hemocytes, respectively. Each of different types of hemocytes specifically expressed the potential marker genes. Based on the responses of shrimp and crayfish to the infection of white spot syndrome virus (WSSV) and the challenge of lipopolysaccharide (LPS), four types of immune hemocytes of crustaceans were classified, including semi-granular hemocytes involved in antimicrobial peptide production, granular hemocytes responsible for the production of antimicrobial peptides, hemocytes related to cell proliferation and hemocytes in immunity-activated state. Therefore, our study provided the first classification of crustacean hemocytes as well as of immune hemocytes of crustaceans at the single-cell resolution, which would be helpful to understand the innate immunity of invertebrates.

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity

BackgroundMosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated.MethodsLarvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR.ResultsPropagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF.ConclusionBy 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.Graphical

Haemocyte characterization in invasive Carcinus maenas (Linnaeus, 1758) (Decapoda, Portunidae) from unpolluted vs polluted sites in Argentina

Abstract Pollution, biological invasions and climate change pose the greatest threats to biodiversity worldwide. Few studies explore the interlink between invasive species and local pollution sites. This study describes the type and cytochemical properties of haemocytes from invasive Carcinus maenas on the southwestern Atlantic coast (Argentina). Moreover, the morphology of haemocytes and the total haemocyte count (THC) as well as the differential haemocyte count (DHC) of crabs sampled at clean or unpolluted and polluted sites were compared, to determine whether environmental stresses were evident in their haemocytes. Our results revealed two groups or subpopulations of haemocytes: hyalinocytes, as well as granulocytes and semi-granulocytes. The type of haemocyte from crabs from both sites showed no differences in their morphology. For cytochemical assays, only neutral red dye showed a more intense staining of crab haemocytes at the polluted site. All haemocytes were positive to Periodic Acid-Schiff and Sudan Black. THC of crabs from the clean site were significantly higher, as well as having a significantly higher proportion of hyalinocytes and a lower proportion of granular cells. This study provides a baseline immunological assessment for C. maenas haemocytes, suggesting a differential haemocytic response to environmental stress.

Metabolic, antioxidant, and immune response of broodstock males ofPenaeus brasiliensisLatreille, 1817 fed diets supplemented by vitamin C

AbstractControlling oxidative stress in shrimps under culture conditions is of vital importance to ensure that males have an optimal physiological status. The use of diets supplemented with vitamins are widely used for this purpose. We evaluated the effects of vitamin C on the metabolic, antioxidant, and immune responses of Penaeus brasiliensisLatreille, 1817 broodstock males under culture conditions. A formulated diet was used as a control (16 mg kg–1 of total ascorbic acid (vitamin C) content) and three diets were prepared with three vitamin C levels: 322 mg kg–1 (Diet A), 628 mg kg–1 (Diet B), and 934 mg kg–1 (Diet C). Triglycerides, cholesterol, glucose, and prophenoloxidase, total proteins, and quantity and type of hemocytes in the hemolymph were determined. We also quantified the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx) and lipid peroxidation in the reproductive tract. Differentially expressed genes of the immune system, namely those of α-2 macroglobulin (α2M), peneidin (Pen), hemocyanin, prophenoloxidase, and the genes encoding the antioxidant enzymes SOD, CAT, and Gpx were quantified in the tract. Sperm quantity and quality were recorded. In males fed Diet C, the lowest cholesterol values and triglycerides obtained in the hemolymph were not significantly different from those fed the control diet (P &amp;lt; 0.05); those fed Diet C showed the highest amount of hyaline hemocytes, the lowest CAT enzyme activity, and the highest Gpx activity. The immune system genes only showed differences between α2M and Pen; sperm quantity and quality were not affected by diet. The results indicated that Diet C (13.9 g kg–1 vitamin C) was effective for the maintenance of P. brasiliensis broodstock males because it controlled cholesterol and triglyceride levels; there was less CAT activity and less activation of the immune system mediated by hemocytes.

The Effects of Environmental Variables and Morphometry on Hemocyte Structure in the Hemolymph of Portunid Crab, Carcinus aestuarii Nardo, 1847 in Çardak Lagoon (Çanakkale Strait)

This study aims to determine the relationship between hemocyte count and hemocyte types with environmental variables and morphometric measurements in Carcinus aestuarii. A total of 240 crabs were seasonally collected in Çardak Lagoon June 2018, October 2018, February 2019, and May 2019. The average carapace length (CL) was 44.66±0.44 mm, the mean carapace width (CW) was 37.71±0.38 mm and the mean weight was 28.49±0.88 g. Crab specimens were anesthetized on ice for 10 min, and 500 µL of hemolymph was collected from the legs of each crab using a 1-mL plastic syringe. After sampling, hemolymph samples were mixed with the anticoagulant, which was applied to a Thoma slide and examined under a microscope at X40 magnification. The total hemocyte count (THC) was calculated as 13.91±8.08x106 (cells/mm3). Differential hemocyte count (DHC) 53.25±0.6% for hyalinocyte, 26.15±0.23% for semi-granulocyte, and 20.6±0.06% for granulocyte and 3 different cell types were observed. The most dominant cell type is hyalinocyte cells.

The involvement of hypoxia inducible factor-1α on the proportion of three types of haemocytes in Chinese mitten crab under hypoxia stress

Hypoxia triggers diverse cell physiological processes, and the hypoxia inducible factors (HIFs) are a family of heterodimeric transcription factors that function as master regulators to respond to hypoxia in different cells. However, the knowledge about the hypoxic responses especially cell alteration mediated by HIFs under hypoxia stress is still limited in crustaceans. In the present study, a hypoxia-inducible factor-1α (HIF-1α) gene was identified (designed as EsHIF-1α). The relative mRNA expression level of EsHIF-1α was highest in hyalinocytes and lowest in granulocytes among three types of haemocytes in crabs. Hypoxia could significantly increase the EsHIF-1α protein expression level in haemocytes. Meanwhile, the proportion of hyalinocytes began to increase from 3 h post hypoxia treatment, and reached the highest level at 24 h. However, the opposite variation in proportion of granulocytes was observed under hypoxia stress. Further investigation showed that the inhibition of EsHIF-1α induced by KC7F2 (HIF-1α inhibitor) could lead to the significant decrease in the proportion of hyalinocytes under hypoxia stress, and also resulted in an increase of granulocytes proportion. While, after EsHIF-1α was activated by IOX4 (HIF-1α activator), the proportion of hyalinocytes was significantly up-regulated and the proportion of granulocytes was significantly down-regulated under post hypoxia treatment. These results collectively suggested that EsHIF-1α was involved in the regulation of proportion of three types of haemocytes induced by hypoxia stress, which provided vital insight into the understanding of the crosstalk between hypoxia and cell development in invertebrates.

Classification and characterization of immune haemocytes in the larvae of the Indian fritillary, Papilio hyperbius (Lepidopetra: Nymphalidae)

In this study, haemocytes present in Papilio hyperbius Linnaeus were identified and characterized. Six different types of haemocyte were recorded in the haemocoel of this species of insect: prohaemocytes, plasmatocytes, granulocytes, spherulocytes, adipohaemocytes and oenocytoids. Of these the granulocytes were found to be responsible for cell-mediated immune responses such as phagocytosis. Granulocytes that were exposed to immunity inducers (carboxylate-modified polystyrene latex beads [CLBs] and Escherichia coli) had fan-like or pod-like structures on their cell membranes. The lysosomes in granulocytes were activated 2 h after injection with E. coli and after 12 h, all granulocytes exhibited highly activated lysosomes. After 24 and 48 h, the lysosome activity in granulocytes decreased. Transmission electron microscopy revealed that phagocytosis, which was mediated by granulocytes in the early hours of the E. coli infection, led to the formation of one phagosome for one E. coli within the cytosol. Moreover, as time passed, endosomes or lysosomes of different size developed. Subsequently, the phagosomes and lysosomes fused and E. coli were eliminated. After this series of immune responses, the nuclei of the granulocytes were indistinct and their cellular activity decreased. Hence, as old immune cells were replaced by new ones, active and healthy immune haemocytes were presumed to be maintained in the hemocoel.

Routine development of long-term primary cell culture and finite cell line from the hemolymph of greasyback shrimp (Metapenaeus ensis) and virus susceptibility

Long-term and continuous shrimp cell cultures can offer an indispensable tool for studies on the isolation, multiplication and pathogenic mechanism of shrimp causative viruses at cellular and molecular levels. However, they are hard to be developed routinely and continuous shrimp cell line is still unavailable. This can be attributed to a great degree to the unsuitability of medium used. In this study, we have successfully developed a long-term culture condition for the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. Using the newly developed gelatin-containing optimal basic medium (SBM-12), MeH cell monolayer could be routinely developed and maintained healthily beyond 275 days. In the novel optimal growth medium (SGM-10), the proliferation potentials of MeH cells have been markedly improved, evidenced by the presence of dividing cells in the primarily cultured 30-day hemocytes, suggesting that the mitosis-arrest of MeH cells has been tentatively broken. It was also found that among the 4 types of hemocytes (granulocyte, semi-granulocyte, hyalinocyte and prohaemocyte), the hyalinocytes could not survive after a long-term in vitro culture, but the prohaemocytes could survive well and thus could serve as a good cell source. The addition of gelatin could significantly alter the attachment and cellular morphology of MeH cells and result in a loose attachment and round shape, which made it easy for the cells to be passaged by physical flushing for up to 32 times and a finite shrimp cell line can be developed routinely. Virus susceptibility test showed that the MeH cells cultured in the optimal growth medium were sensitive to shrimp covert mortality nodavirus (CMNV), and obvious cytopathic effects and viral multiplication were detected in the infected cells. In a word, the success in the routine development of long-term primary cell culture and finite cell line from shrimps in this study will greatly facilitate the establishment of shrimp continuous cell lines and studies on shrimp viruses.

Morphofunctional characterization of hemocytes in black soldier fly larvae.

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H.illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.

Evaluation of farnesol-induced changes in the haemocyte pattern of red cotton bug Dysdercus koenigii Fabricius (Heteroptera: Pyrrhocoridae)

BackgroundDysdercus koenigii Fabricius is a polyphagous agricultural pest of paramount importance. Farnesol is a plant-derived open-chain aliphatic sesquiterpenoid alcohol. It is an intermediate in the metabolic pathway of juvenile hormone biosynthesis. The effects of Farnesol on haemocytes of fifth instar nymphs of Dysdercus koenigii were assessed in the present investigation. The early fifth instar nymphs were treated with doses of 0.05 μL, 0.1 μL and 0.2 μL Farnesol topically and day wise change in the total haemocytes was reported. Different types of haemocytes were identified in the haemolymph based on their morphological characteristics. Difference in day wise distribution pattern of haemocytes was evaluated in relation to dose of treatment.ResultsIn normal fifth instar nymphs, age-related variation in the haemocyte count was evident; the haemocyte number increased up to four days and subsequently decreased till six-day when the nymph moulted. The results indicate that Farnesol had an adverse effect on the total number of haemocytes; in treated nymphs the haemocyte count decreased in a dose-dependent manner. The decrease was most prominent in the nymphs treated with a dose of 0.2 μL Farnesol. Based on the morphological characteristics, six different types of haemocytes, viz. prohaemocyte, plasmatocyte, adipohaemocyte, granulocytes, oenocytoids and disintegrated cells were identified in the haemolymph. The percent composition of different haemocytes showed variation depending on the age of the fifth instar nymph and the dose of treatment. In control, the percentages of plasmatocytes decreased up to day 5 and then increased up to 7 days. The percentage of adipohaemocytes increased throughout the nymphal period of the fifth instar, except for a small dip on the fourth day. Granulocytes and oenocytoids showed no clear trend. In Farnesol-treated nymphs, an increase in percent of prohaemocytes was observed in all the treatments. Dose-dependent effects were more prominent after day 5. An increase in plasmatocytes was seen after day 3. A distinct dose-related decrease was reported in adipohaemocytes on all days. Percent of disintegrated cells also showed an increase in all the tested doses of Farnesol on all the days. Granulocytes and oenocytoids showed no definitive trend of change.ConclusionsFarnesol is a plant sesquiterpenoid and acts as a precursor in juvenile hormone biosynthesis. The application of Farnesol exogenously may disturb haemocyte patterns which may challenge the immune response of insects. Therefore, Farnesol can potentially be used as an alternative approach in pest management.

EDTA-Na&lt;sub&gt;2&lt;/sub&gt; - anticoagulant for hemolymph of australian red claw crayfish (cherax quadricarinatus)

Purpose: selection of anticoagulants based on EDTA-Na2 and methods of their use for working with the hemolymph of the Australian red claw crayfish (C. quadricarinatus)Materials and methods. For the study, different-sized males and females of the australian red-clawed crayfish (Cherax quadricarinatus (Von Martens, 1868)) were used. EDTA-Na2 was used as an anticoagulant, the concentrationof which was 4%. A 2 ml syringe with a 23G needle for hemolymph removal was pre-washed with anticoagulant remaining in the needle and the needle cone (about 1/3 of the volume of the needle cone was filled with a solution). The experiment is presented in the following series: 1. differences between the total hemocytes number (THC) and the proportion of granulocytes in native hemolymph and treated with a small amount of EDTA-Na2; 2. differences in the content of total hemolymph protein (THP) in native and treated with a small amount of EDTA-Na2 hemolymph; 3. changes in THC and the proportion of granulocytes in hemolymph treated with a small amount the amount of EDTA-Na2 immediately after sampling and a day later. To carry out the work, samples of 20 individuals were used, while for the first two series, two samples of hemolymph were taken from each, and one from the third. Syringes with hemolymph treated with anticoagulant were stored in the refrigerator at a temperature of 8.5 °C. Additionally, similar studies were carried out on differences in the THC and proportion of granulocytes in the hemolymph immediately after sampling with treatment with a multicomponent anticoagulant consisting of 4 g of EDTA-Na2, 3 g of sodium citrate, 2 g of glucose and 1 g of NaCl per 100 ml of distilled water. Hemolymph was taken with a syringe from the ventral sinus. Three indicators were determined: the total number of hemocytes (THC), the proportion of granulocytes and the total protein content (THC). THC and proportion of granulocytes was determined in the Goryaev chamber under a light microscope. THP was determined by the refractometric method.Results. The study revealed significant differences in the proportion of granulocytes (p&lt;0.05), which are 32% more in the anticoagulant treated than in the native hemolymph, which can be explained by the uniform distribution of all types of hemocytes in the sample. There were no significant differences in the remaining indicators of all series. Studies of hemolymph treated with a multicomponent anticoagulant showed a low level of hemocyte preservation (p&lt;0.05) and the proportion of granulocytes compared to the indicators immediately after selection, therefore, they are not reflected in the work. Conclusion. The data obtained indicate that there is a possibility of using four percent EDTA-Na2 when working with hemolymph. The anticoagulant prevents the formation of gel and the rapid destruction of hemocytes, promotes the uniform distribution of cells in the Goryaev chamber and allows the use of a refractometer in determining the total blood protein. The use of an anticoagulant contributes to the preservation of hemocytes during the day, at a level that allows the use of EDTA-Na2 in practice. At the same time, work should continue on the development of methods for storing and transporting blood treated with an anticoagulant.

Characterization of hemocytes and hematopoietic cells of a freshwater crayfish based on single-cell transcriptome analysis

SummaryCrustaceans constitute a species-rich and ecologically important animal group, and their circulating blood cells (hemocytes) are of critical importance in immunity as key players in pathogen recognition, phagocytosis, melanization, and antimicrobial defense. To gain a better understanding of the immune responses to different pathogens, it is crucial that we identify different hemocyte subpopulations with different functions and gain a better understanding of how these cells are formed. Here, we performed single-cell RNA sequencing of isolated hematopoietic tissue (HPT) cells and hemocytes from the crayfish Pacifastacus leniusculus to identify hitherto undescribed hemocyte types in the circulation and show that the circulating cells are more diversified than previously recognized. In addition, we discovered cell populations in the HPT with clear precursor characteristics as well as cells involved in iron homeostasis, representing a previously undiscovered cell type. These findings may improve our understanding of hematopoietic stem cell regulation in crustaceans and other animals.

Characterization of mussel ( Perna canaliculus ) haemocytes and their phagocytic activity across seasons

Perna canaliculus is endemic to New Zealand and is the top shellfish export product. However, the growth of this industry is being adversely affected by summer mortality events. To assess these health threats, differential haemocyte counts (DHC) were performed on haemolymph smears stained with Giemsa, and in vitro phagocytosis activity assays were applied in P. canaliculus for several months covering three seasons (winter, spring and summer). A new optimized classification scheme was developed for P. canaliculus haemocytes where 55% eosinophilic and 27.2% basophilic granulocytes were identified. Eosinophilic granulocytes were classified as dense, semi and small semi, and these new categories were reported herein for the first time in this species. A new haemocyte type (mixed granulocytes), which contains both acidophilic and basophilic granules, is proposed for P. canaliculus. The phagocytosis percentages were significantly affected by incubation time, indicating an increase in zymosan particle uptake from 18.42 ± 1.7% after 30 min, 32.08 ± 3.1% after 60 min and 44.74 ± 3.5% (maximum) after 120 min of incubation. The lowest phagocytosis was observed in the winter season and the highest phagocytosis in summer. This study findings provide a better understanding of the immune function of P. canaliculus haemocytes and serve as a reference for further investigations on the impacts of seasonal variations on haemocyte activity and phagocytosis.

Does Bacillus thuringiensis Affect the Stress and Immune Responses of Rhynchophorus ferrugineus Larvae, Females, and Males in the Same Way?

Simple SummaryRhynchophorus ferrugineus is a destructive quarantine pest of palm trees, now widely distributed. Although broad-spectrum insecticides are often used to protect palm against R. ferrugineus, there is increasing concern about their effects on the environment and human health, especially where palm trees are located in urban areas. As an environmentally friendly entomopathogen, Bacillus thuringiensis (Bt) has been widely used to prevent other pest infestations. Although Bt products are the most sold bio-insecticides, there are still many interesting features to be investigated in the relationship of Bt and its hosts. We investigated the effect of Bt on larvae, females, and males. This research yielded experimental evidence of significant mortality and significant effects on immune system and stress answer. Within a few hours, stress due to Bt infection was detected in the hemocytes and in the brain providing better insights into the insect-pathogen interaction and highlighting the potential use of Bt in R. ferrugineus management.Bacillus thuringiensis (Bt) is considered a potentially useful entomopathogen against red palm weevil (RPW) Rhynchophorus ferrugineus. We compared the effects of Bt on mature larvae, females, and males. The pathogenicity of Bt was evaluated, estimating: Median Lethal Dose (LD50), Median Lethal Time (LT50), Total Hemocyte Count (THC), and Differential Hemocyte Counts (DHC), and the expression of the stress protein Heat Shock Protein 70 (Hsp 70) in hemocytes and the brain. Mortality exhibited a positive trend with the dosage and duration of exposure to Bt. Larvae were more susceptible than adults, and the LD50 of females was almost double the value of that of the larvae. LT50 value was higher for females than for males and larvae. Treatment with sub-lethal doses of Bt induced a decrease in THC in larvae, females, and males. In treated larvae, plasmatocytes decreased, while oenocytes and spherulocytes increased. In treated females, all types of hemocytes decreased, while in males the number of plasmatocytes decreased and granulocytes increased. We also registered the stress response directly on hemocytes showing that, already at 3 h after eating Bt, the expression of the stress protein Hsp 70 was modulated. This effect was also observed in brain tissue at 6 h after treatment. The results confirm that Bt treatment induces a pathogenic state in larvae and adults of both sexes, with effects after only a few hours from ingestion; however, the effects are different in magnitude and in type of target.

Single-Cell Sequencing Reveals Types of Hepatopancreatic Cells and Haemocytes in Black Tiger Shrimp (Penaeus monodon) and Their Molecular Responses to Ammonia Stress.

The cell types and developmental trajectories of shrimp cells based on the transcriptional level have not been established, and gene expression profile and function at the single-cell level is unclear. We aimed to use scRNA-seq to construct a single-cell resolution transcriptional map of hepatopancreas and haemocytes in shrimp to analyse the molecular mechanisms of the immune response to ammonia nitrogen stress. In the present study, seven cell clusters were successfully identified in each of the two tissues (haemocytes, Hem1-7; hepatopancreas, Hep1-7) based on specifically-expressed marker genes. The developmental starting points of haemocytes and hepatopancreatic cells were Hem2 and Hep1, respectively. We propose that Hem2 has oligopotent potential as the initiation site for haemocyte development and that Hem4 and Hem5, located at the end of development, are the most mature immune cell types in haemocytes. Hep5 and Hep6 were the developing terminal cells of hepatopancreas. The antioxidant system and proPO system of shrimp were activated under ammonia nitrogen stress. A large number of DEGs were involved in oxidative stress, detoxification metabolism, and immune defence. In particular, important response genes such as AMPs, proPO, and GST were not only marker genes for identifying cell groups but also played an important role in shrimp cell differentiation and functional plasticity. By successfully applying 10× Genomics based scRNA-seq to the study of shrimp, the single-cell transcriptional profiles of hepatopancreatic cells and haemocytes of shrimp innate immune responses under ammonia stress were constructed for the first time. This atlas of invertebrate hepatopancreatic cells and haemocytes at single-cell resolution identifies molecular events that underpin shrimp innate immune system responses to stress.

Time-Course of the Innate Immune Response of the Terrestrial Crustacean Porcellio scaber After Injection of a Single Dose of Lipopolysaccharide.

Invertebrates, including crustaceans, rely on cellular and humoral immune responses to protect against extrinsic and intrinsic factors that threaten their integrity. Recently, different immune parameters have been increasingly used as biomarkers of effects of pollutants and environmental change. Here, we describe the dynamics of the innate immune response of the terrestrial crustacean Porcellio scaber to injection of a single dose of lipopolysaccharide (LPS), an important molecular surface component of the outer membrane of Gram-negative bacteria. The aim was to provide a basis for interpretation of change in immune parameters as a result of different challenges, including microplastics and nanoplastics exposure. Changes in total and differential numbers of hemocytes, hemocyte viability, and humoral immune parameters (i.e., phenoloxidase-like activity, nitric oxide levels) were assessed at different times (3, 6, 12, 24, 48 h). An injection of 0.5 μg/μL LPS into the body of P. scaber resulted in a rapid decrease (3 h after LPS injection) in the total number of hemocytes and reduced viability of the hemocytes. This was accompanied by changed proportions of the different hemocyte types, as a decrease in the numbers of semigranulocytes and granulocytes, and a marked increase in the numbers of hyalinocytes. In addition, phenoloxidase-like activity and nitric oxide levels in the hemolymph were increased at 3 h and 6 h, respectively, after the LPS challenge. Forty-eight hours after LPS injection, the immune parameters in the hemolymph of P. scaber had returned to those before the LPS challenge. This suggests that the innate immune system successfully protected P. scaber from the deleterious effects of the LPS challenge. These data indicate the need to consider the dynamics of innate immune responses of P. scaber when effects of infections, pollutants, or environmental changes are studied. We also propose an approach to test the immunocompetence of organisms after different challenges in ecotoxicity studies, based on the dynamics of their immune responses.

Prohemocytes are the main cells infected by dengue virus in Aedes aegypti and Aedes albopictus

BackgroundThe primary disease vectors for dengue virus (DENV) transmission between humans are the mosquitoes Aedes aegypti and Aedes albopictus, with Ae. aegypti population size strongly correlated with DENV outbreaks. When a mosquito is infected with DENV, the virus migrates from the midgut to the salivary glands to complete the transmission cycle. How the virus crosses the hemocoel, resulting in systemic infection, is still unclear however. During viral infection and migration, the innate immune system is activated in defense. As part of cellular-mediated immunity, hemocytes are known to defend against bacteria and Plasmodium infection and may also participate in defending against DENV infection. Hemocytes are categorized into three cell types: prohemocytes, granulocytes, and oenocytoids. Here, we investigated which hemocytes can be infected by DENV and compare hemocyte infection between Ae. aegypti and Ae. albopictus.MethodsHemocytes were collected from Ae. aegypti and Ae. albopictus mosquitoes that were intrathoracically infected with DENV2-GFP. The collected hemocytes were then identified via Giemsa staining and examined microscopically for morphological differences and viral infection.ResultsAll three types of hemocytes were infected by DENV, though the predominantly infected cell type was prohemocytes. In Ae. aegypti, the highest and lowest infection rates at 7 days post infection occurred in prohemocytes and granulocytes, respectively. Prohemocytes were also the primary infection target of DENV in Ae. albopictus, with similar infection rates across the other two hemocyte groups. The ratios of hemocyte composition did not differ significantly between non-infected and infected mosquitoes for either species.ConclusionsIn this study, we showed that prohemocytes were the major type of hemocyte infected by DENV in both Ae. aegypti and Ae. albopictus. The infection rate of prohemocytes in Ae. albopictus was lower than that in Ae. aegypti, which may explain why systemic DENV infection in Ae. albopictus is less efficient than in Ae. aegypti and why Ae. albopictus is less correlated to dengue fever outbreaks. Future work in understanding the mechanisms behind these phenomena may help reduce arbovirus infection prevalence.Graphical

Study of the haemocytic parameters of cockroaches (Periplaneta americana) post stimulation with bacterial spores

Haemocytes and haemolymph are the major mediators of immunity in cockroaches (Periplaneta americana). The six different types of haemocytes reported are prohaemocytes, plasmatocytes, granular haemocytes, oenocytoids, spherules and adipohaemocytes. These cells have their own structural and functional uniqueness. In this study, we injected cockroaches with bacterial spores and compared the number and size of the haemocytes in control and inoculated populations. The number of viable haemocytes in the inoculated population was less than that in control. There was a significant increase in the number and size of plasmatocytes in the immunized cockroaches. The adipohaemocytes also showed increase in size in the inoculated population. Both plasmatocytes and adipohaemocytes exhibit phagocytic activity. Additionally, more production of superoxide anions in the haemocytes of inoculated cockroaches are indicative of increased phagocytosis. We also report a change in protein profile of haemolymph of the injected cockroaches. The heightened activity of few haemocytes and bio-molecules in the immunized cockroaches, hint towards an activated cellular and humoral immune response due to inoculation. Together, these findings re-establish the fact that the invertebrate immune system responds variably to antigenic challenge.