Semigranular Cells Research Articles

Comparison analysis of circulating hemocytes in decapod crustaceans

Hemocytes are the primary immune cells of crustaceans. Few comparison studies have been done among different crustaceans and some key parameters of circulating hemocytes have not been investigated. Here, we compared the circulating hemocytes in six decapod crustaceans, Cherax quadrinatus, Procambarus clarkii, Penaeus vannamei, Penaeus monodon, Eriocheir sinensis, and Scylla paramamosain. Although the hemocytes of different species vary in size, they share common morphological characteristics. Based on their morphological features, circulating hemocytes can be basically classified into granular cells (GCs), semi-granular cells (SGCs), and hyaline cells (HCs). In the six decapods analyzed in this study, the proportion of GCs varied from 10 % to 30 %. P. vannamei, P. monodon, and P. clarkii had fewer GCs in circulation than the other three species. Correspondingly, proliferation was detected only in a small portion of cells in P. vannamei, P. monodon, and P. clarkii under physical conditions. The hemocyte renewal rates for P. clarkii, E. sinensis, and C. quadrinatus were 6.1 %, 5.1 %, and 1.5 % per day, while no steady new hemocyte production was found in S. paramamosain within six days. These data give a general picture of the similarities and differences of circulating hemocytes in decapods and provide a base for an in-depth study of their immune system.

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

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

Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulata

Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 μL of physiological crustacean (PS), while the rest received 10 μL of LPS [0.1 mg.kg−1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.

The effects of dietary black cumin (Nigella sativa) seed meal on immune response of Pacific white shrimp (Penaeus vannamei)

ABSTRACT The black cumin (Nigella sativa) seed (BCS) is known as an immunostimulant herb owing to its enhancing effects on nonspecific immune systems. However, the effectiveness of dietary BCS on shrimp immune systems has not been well known. Therefore, the present study evaluated the effects of the experimental diets containing 0 (control), 5 (BCS5), 15 (BCS15), 30 (BCS30), and 50 (BCS50) g kg−1 (BCS) on immune response of Pacific white shrimp (Penaeus vannamei). The results showed significant decrease in plasma glucose, globulin, ALP activity and hyaline cells and increase of semigranular cells in shrimp fed up to 30 g kg−1 BCS compared to the control group (p < 0.05). Moreover, significant upregulation of α2-macroglobulin (α2-M), integrin β, and peroxinectin (PX) transcripts was observed during the course of the experiment in all treatments (p < 0.05). The transcripts of PX and integrin β showed a significant decrease in BCS fed shrimp after exposure to V. harveyi (p < 0.05). In conclusion, supplementation of BCS in the diet of shrimp for 90 days and after exposure to V. harveyi could significantly affect some immunological indices and the expression of some immunity related biomarkers.

Exploring the combined interplays: Effects of cypermethrin and microplastic exposure on the survival and antioxidant physiology of Astacus leptodactylus

Plastic waste and micro/nanoplastic particles pose a significant global environmental challenge, along with concerns surrounding certain pesticides' impact on aquatic organisms. This study investigated the effects of microplastic particles (MPPs) and cypermethrin (CYP) on crayfish, focusing on biochemical indices, lipid peroxidation, oxidative stress, hematological changes, and histopathological damage. After determining the LC50–96 h value (4.162 μg/L), crayfish were exposed to sub-lethal concentrations of CYP (1.00 ppb (20%) and 2.00 ppb (50%)) and fed a diet containing 100 mg/kg MPPs for 60 days. Hemolymph transfusion and histopathological examinations of the hepatopancreas were conducted. The results showed significant alterations in crayfish. Total protein levels decreased, indicating protein breakdown to counteract contaminants, while total cholesterol and triglyceride levels declined, suggesting impaired metabolism. Glucose levels increased in response to chemical stress. The decline in total antioxidant capacity highlighted the impact of prolonged xenobiotic exposure and oxidative stress, while increased CAT, SOD, and MDA activities helped mitigate oxidative stress and maintain cellular homeostasis. The elevated total hemocyte count, particularly in semi-granular cells, suggests their active involvement in the detoxification process. Further research is needed to fully understand the implications of these effects.

In Silico and In Vivo Potential of Fraction Red Betel Leaf as an Immunostimulant Agent in White-leg Shrimp

Production of white-leg shrimp (Litopenaeus vannamei) in aquaculture with advanced technology ultimately results in high mortality rates in cultivation. Infectious diseases, including Vibrio spp., can cause mortality with necrosis symptoms in the white-leg shrimp hepatopancreas. Disease prevention through enhancing immunity is highly effective in controlling diseases in shrimps. The current study aimed to obtain information on the compounds present in the fraction of Piper (P.) crocatum as an immunostimulant agent used in silico. The current study investigated the absorption, distribution, metabolism, excretion, and toxicity (ADME/T), and determined the optimal fraction dosage of P. crocatum when injected as an immunostimulant substance. In this study, in silico analysis was conducted by accessing several servers, while the shrimp’s immune response was evaluated using a completely randomized design experiment with four treatments (10 individuals/container) and three replications, including 0 (control), 0.5 µg/g, 1 µg/g, and 1.5 µg/g. The shrimp’s immunity was examined by injecting the P. crocatum fraction initially, followed by a second injection 24 hours later. Shrimp hemolymph was collected before the injection of the P. crocatum fraction and 24 hours after the injection. Hemolymph was collected at both time intervals to assess total hemocyte count (THC), differential hemocyte count (DHC), and phenoloxidase (PO) as the immune response of shrimp before and after administration of P. crocatum fraction. Two compounds were confirmed immunostimulant agents in a fraction of P. crocatum, 2-Amino-1,3,4-octadecanetriol, and erucamide. The immune response values for THC (14.17 ± 2.45 × 106 cells mL-1), DHC hyaline (53 ± 4.5%), semi-granular cells (52 ± 4.0%), and granular cells (43 ± 40%), and PO (0.112 ± 0.016 units/λ=490) at a concentration of 1.5 µg/g showed a significant increase in number and percentage compared to the control. These results indicate the presence of two compounds in fraction one P. crocatum, as candidates for immunostimulant agents. Administration of 1.5 µg/g of a fraction of P. crocatum is the appropriate dose as an immunostimulant agent when administered via injection method for white-leg shrimp.

Dietary Effects of Lactobacillus plantarum Combined with Galactooligosaccharide on Immunological and Biochemical Parameters, Gut Microbiota, Digestive Enzyme Activity, Body Composition, and Stress Resistance in Narrow-Clawed Crayfish, Pontastacus leptodactylus (Eschscholtz, 1823)

The effects of galactooligosaccharide (GOS) and varying levels of Lactobacillus plantarum (LP) on some physiological parameters as well as air exposure stress resistance in clawed crayfish (Pontastacus leptodactylus) were investigated. During a 97-day trial, 216 crayfish were fed with four experimental diets (in triplicates) including the control diet (without GOS and LP), GLP7 (GOS 2%+LP 107 CFU.g-1 diet), GLP8 (GOS 2%+LP 108 CFU.g-1 diet), and GLP9 (GOS 2%+LP 109 CFU.g-1 diet). At the end of the trial, 26% higher amounts of the total hemocyte count (THC) and semigranular cells (SGC) and 27% higher hyaline cells (HC) were observed in GLP7. A significant improvement in lysozyme value was observed in GLP7 and GLP8. Moreover, superoxide dismutase was significantly higher in GLP9, whereas the catalase (CAT) activity did not change in the experimental groups. Unlikely, plasma glucose levels decreased in all the GLP treatments. In terms of intestinal microbiota, autochthonous lactic acid bacteria (LAB) remarkably increased in all the GLP-supplemented groups, while total autochthonous intestinal heterotrophic bacteria counts (TVC) did not change in GLP-supplemented groups. However, 14 days after switching to the basal diet, no reduction was detected in TVC, but LAB levels decreased in GLP7 and GLP9 treatments. Dietary administration of GLP could beneficially modulate digestive enzyme activity, including protease, lipase, and amylase. Furthermore, higher lipid and gross energy were observed in GLP9. However, GLP-supplemented diets could not improve growth performance parameters. After 24 h subjecting to air exposure stress, although no mortality was detected, crayfish fed GLP-supplemented diets were healthier through enhanced THC, SGC, and HC levels in GLP7 and GLP9 and CAT activity in GLP7 and GLP8. These results proved the positive impacts of dietary supplementation with combined GOS and LP, with GLP8 and GLP9 as optimum diets, on immunochemical parameters, intestinal microbiota, digestive enzyme activity, and stress resistance in P. leptodactylus.

Comparison of Immune Response of Litopenaeus vannamei Shrimp Naturally Infected with Vibrio Species, and after Being Fed with Florfenicol

The outbreaks caused by Vibrio spp. are a notable threat to the potential growth of the economy of penaeid culture, which is still controlled by the administration of antibiotics. At first, the infected group was subjected to phenotypic bacteriological examination with subsequent molecular identification via 16S rRNA gene sequencing, which confirmed four strains of Vibrio spp., V. atlanticus, V. natriegens, V. alginolyticus, and V. harveyi, from moribund-infected shrimp during mortality events in an Egyptian hatchery. To better understand the defense mechanism of the most effective antibiotic against Vibrio strains, the immune responses were compared and evaluated in infected Litopenaeus vannamei broodstock after being fed 5 mg kg−1 of florfenicol antibiotic, which was first determined through in vitro antibiogram tests. Therefore, our study aimed to determine the immune response of L. vannamei during Vibrio spp. infection in Egyptian hatcheries and after antibiotic medication. The parameters assessed were the total and differential hemocyte count (THC), granular cells (GC), semi-granular cells (SGC), and hyaline cells (HC). As well as the metabolic and immune enzymes: alanine aminotransferases (ALT), aspartate aminotransferases (AST), alkaline phosphatase (ALP), acid phosphatase (ACP), and lysozyme activity; an antioxidant index, such as superoxide dismutase (SOD) and glutathione (GSH); a phagocytic assay; changes in reactive oxygen species (ROS); and bactericidal activity in the hemolymph of the control, infected, and treated groups. Further evaluation of the mRNA expression levels of the prophenoloxidase (LvproPO), toll-like receptor 1 (LvToll1), and haemocyanin (LvHc) genes were performed in the hepatopancreas of the same groups. A significant drop in the THC, GC, SGC, and HC counts, as well as lysozyme and bactericidal activities, phagocytic assay, ROS, SOD, and GSH index, were represented in infected shrimp compared to control shrimp; however, a marked increase in the activity of ALT, AST, ALP, and ACP was observed. These activities were significantly restored in the treated shrimp compared to the infected shrimp. Nevertheless, no significant changes were noted in the transcriptional levels of the LvproPO and LvToll1 genes in the treated shrimp when compared to the infected shrimp; however, a significant suppression of the LvHc gene was noted. Our study aimed to determine the immune response of L. vannamei during Vibrio spp. infection in Egyptian hatcheries and after antibiotic medication. We concluded that florfenicol in medicated feed could be effective in controlling vibriosis and ameliorating the immune response of shrimp.

Physiological response of freshwater crayfish, Astacus leptodactylus exposed to polyethylene microplastics at different temperature

Not long after plastic-made material became an inseparable part of our lives, microplastics (MPs) found their way into ecosystems. Aquatic organisms are one of the groups impacted by man-made materials and plastics; however, the varied effects of MPs on these organisms have yet to be fully understood. Therefore, to clarify this issue, 288 freshwater crayfish (Astacus leptodactylus) were assigned to eight experimental groups (2 × 4 factorial design) and exposed to 0, 25, 50, and 100 mg polyethylene microplastics (PE-MPs) per kg of food at 17 and 22 °C for 30 days. Then samples were taken from hemolymph and hepatopancreas to measure biochemical parameters, hematology, and oxidative stress. The aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, and catalase activities increased significantly in crayfish exposed to PE-MPs, while the phenoxy-peroxidase, gamma-glutamyl peptidase, and lysozyme activities decreased. Glucose and malondialdehyde levels in crayfish exposed to PE-MPs were significantly higher than in the control groups. However, triglyceride, cholesterol, and total protein levels decreased significantly. The results showed that the increase in temperature significantly affected the activity of hemolymph enzymes, glucose, triglyceride, and cholesterol contents. The semi-granular cells, hyaline cells, granular cell percentages, and total hemocytes increased significantly with the PE-MPs exposure. Temperature also had a significant effect on the hematological indicators. Overall, the results showed that temperature variations could synergistically affect the changes induced by PE-MPs in biochemical parameters, immunity, oxidative stress, and the number of hemocytes.

Utilization of Jatropha Leaf (Jatropa curcas) Extract as Immune System Enhancer of Vannamei Shrimp (Litopenaeus vannamei) Infected with Vibrio parahaemolyticus

The problem arises in vanamei shrimp cultivation is the emergence of diseases, such as vibriosis caused by Vibrio parahaemolyticus. Jatropha leaf extract can be used as an immunostimulant because it contains bioactive compounds namely flavonoid, tannins, saponins, and alkaloids. This research used a completely randomized design (CRD) with 5 treatments with 3 replications. The treatments used were; P1 (feed without extract and bacterial infection); P2 (feed without extract and NaCl injection); P3 : Feed + 1% + bacterial infection; P4 : Feed + 2% extract from feed and bacterial infection, P5 : Feed + 3% extract of feed and bacterial infection. Based on the results of the study, the administration of Jatropha leaf extract was able to increase the highest SR value of P2 by 88.3%, the highest THC value of P5 at 24.46 x 106 cells/ml, DHC values ​​such as hyaline cells ranged from 36.67-71%, granular cells ranged from 17-38.57%, and semigranular cells ranged from 12-24.67%, the highest AF value in P5 was 77.25%, and the lowest TBC value was at P2 of 6 x 108 CFU/ml, the lowest TVC value was at P2 of 0.56 x 106 CFU/ml. The higher the dose of extract given, the higher the THC and AF, besides that, Jatropha leaves were also able to increase the SR value and suppress the growth of total bacteria and total Vibrio in shrimp intestines.

ScRNA-seq analysis reveals toxicity mechanisms in shrimp hemocytes subjected to nitrite stress

In shrimp, hemocytes play an important role in detoxification and immune defense, and are where nitrite accumulates during exposure to this toxic environmental pollutant. However, the heterogeneity mechanisms of toxicity have not been reported under nitrite expose in shrimp. Here, we used single-cell RNA-seq to resolve 24,000 cells, which the responses of different cell populations of hemocytes under nitrite exposure in Penaeus vannamei. We identified 394 specific nitrite-responsive genes in 9 clusters of hemocytes, and found heterogeneity in the nitrite response of the three subpopulations of hemocytes (hyaline, semi-granular and granular cells). In hyaline, the response appeared modest, whereas nitrite-related dysregulation of metabolic processes in granular and semi-granular was pronounced. Ammonia nitrogen will rapidly accumulate in hemocytes of shrimp under nitrite stress. In semi-granular, excessive ammonia will interfere with oxidative phosphorylation and antioxidant system, thus inducing the production of reactive oxygen species. In granular, the abnormality of urea cycle caused by ammonia accumulation is the main toxic factor, which by inhibits arginase and arginine kinase. Collectively, our data provide a single-cell atlas for the dissection of shrimp hemocyte complexity, and reveal the toxicity mechanisms associated with nitrite exposure.

THE EFFECT OF EM4 PROBIOTIC ADMINISTRATION FOR GROWTH AND SURVIVAL OF VANAME SHRIMP (Litopenaeus vannamei)

Vannamei shrimp is a shrimp that has high economic value in Indonesia. The poor aquatic environment can reduce shrimp growth rates and stress and cause death, and efforts to improve water quality by giving Probiotics EM4. Probiotic EM4 contains beneficial bacteria that can improve shrimp's digestive system, decompose organic matter in the waters, control the growth of pathogenic bacteria, and improve water quality. The experimental parameters included total hemocyte count (THC), differential hemocyte count (DHC) consisting of hyaline cells, granule cells, and semi-granule cells, and phagocytic activity (AF). The study was carried out for 69 days using vanamei shrimp with PL 30. This study used four treatments and three replications, namely treatment P0 (without giving probiotic EM4), P1 (giving probiotic EM4 at a dose of 0.5 ml/L), P2 (giving probiotic EM4 at a dose of 1.5 ml/ L), P3 (giving probiotic EM4 dose of 2.5 ml/L). The study's results proved that the administration of probiotic EM4 on the maintenance medium had a significant effect (P&lt;0.05) on THC, DHC, and AF. At THC, the highest value was at P3 with a value of 19.44 x 10 6 cells/ml, and the lowest was at P0 with a value of 11.01 x 10 6 cells/ml, then the DHC values such as hyaline cells ranged from 51.34 – 65.81%, granule cells ranged from 21.33 – 30.4%, semi-granular cells ranged from 12.84 – 18.24%. And then for the highest AF value at P3 with a value of 67.97% and the lowest P0 with a value of 49.34%

Effects of Dietary Pectin and Lactobacillus salivarius ATCC 11741 on Growth Performance, Immunocompetence, Gut Microbiota, Antioxidant Capacity, and Disease Resistance in Narrow-Clawed Crayfish, Postantacus leptodactylus.

The present study was conducted to clarify the effects of Lactobacillus salivarius (LS) ATCC 11741 and pectin (PE) on growth performance, digestive enzymes activity, gut microbiota composition, immune parameters, antioxidant defense as well as disease resistance against Aeromonas hydrophila in narrow-clawed crayfish, Postantacus leptodactylus. During 18 weeks trial feeding, 525 narrow-clawed crayfish juvenile (8.07 ± 0.1 g) fed with seven experimental diets including control (basal diet), LS1 (1 × 107 CFU/g), LS2 (1 × 109 CFU/g), PE1 (5 g/kg), PE2 (10 g/kg), LS1PE1 (1 × 107 CFU/g +5 g/kg), and LS2PE2 (1 × 109 CFU/g +10 g/kg). After 18 weeks, growth parameters (final weight, weight gain, and specific growth rate) and feed conversion rate were significantly improved in all treatments (P < 0.05). Besides, diets incorporated with LS1PE1 and LS2PE2 significantly increased the activity of amylase and protease enzymes compared to LS1, LS2, and control groups (P < 0.05). Microbiological analyses revealed that the total heterotrophic bacteria count (TVC) and lactic acid bacteria (LAB) of narrow-clawed crayfish fed diets containing LS1, LS2, LS1PE1, and LS2PE2 were higher than control group. The highest total haemocyte count (THC), large-granular (LGC) and semigranular cells (SGC) count, and hyaline count (HC) was obtained in LS1PE1 (P < 0.05). Similarly, higher immunity activity (lysozyme (LYZ), phenoloxidase (PO), nitroxidesynthetase (NOs), and alkaline phosphatase (AKP)) observed in the LS1PE1 treatment compared to the control group (P < 0.05). The glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity remarkably enhanced in LS1PE1 and LS2PE2, while malondialdehyde (MDA) content reduced in these two treatments. In addition, specimens belonging to LS1, LS2, PE2, LS1PE1, and LS2PE2 groups presented higher resistance against A. hydrophila compared to the control group. In conclusion, feeding narrow-clawed crayfish with synbiotic had higher efficiency on growth parameters, immunocompetence, and disease resistance compared to single consumption of prebiotics and probiotics.

Performance of pacific white leg shrimp fed an open-source marine resource-free diet under commercial conditions

Pacific white leg shrimp were stocked (150 PL m-3) into one of four plastic-lined 1000 m3 (0.1 ha) ponds. Ponds were randomly fed one of two diets in duplicate: a commercial shrimp feed (Grobest® Vista Eco), or an open formula, experimental, fishmeal and fish oil-free diet (F3). Shrimp were fed by hand and automatic feeders using commercial feed tables for 56 days. Sub-samples of shrimp (n = 50) were weighed weekly from each pond to evaluate growth and adjust feeding rates. Following harvest, no differences were observed between diets for survival. Combined pond biomass was 2045.0±45.96 kg (S.D.) and 2302.5±28.99 kg for commercial and F3 shrimp respectively. FCR were 1.15±0.00 and 1.03±0.01 (P< 0.05). Individual F3 fed shrimp were significantly (P< 0.05) heavier at trial termination with animals fed the commercial diet weighing 18.03±0.24 g versus 23.25±0.84 g (P< 0.05). Total hemocyte counts were 33.26±0.46 and 33.88±0.72, that for semi-granular cells 20.19±1.65 and 18.72±0.81, granular cells, 15.20±1.39 and 15.56±0.15, hyaline cells, 64.62± 3.01 and 65.72±0.96, and phenoloxidase activity, 56.87±6.12 and 50.29±0.55, for commercial and F3 fed shrimp respectively. A basic economic analysis indicated increased profitability for the F3 fed shrimp (US$7362.43 versus US$10665.62) with a return on investment (ROI) of 64.5% for the commercial feed and 89.8% for the F3 ponds. There were no differences between samples for proximate composition, cooked tail color as assessed by a BASF Salmon Color Fan or texture, as determined by a texture analyzer. Results from this trial unconditionally demonstrate the practicality of cultivating white leg shrimp using marine resource-free-based diets without impacting growth performance or consumer acceptability, while returning increasing ROI.

Depuration effect on the total hemocytes count and heavy metals concentration in freshwater crayfish, Procambarus clarkii

One of the most important aquatic macro-invertebrates is Procambarus clarkii. This study was designed to evaluate the effect of depuration for 21 days on the total hemocytes count and heavy metals (Co, Cr, Pb and Hg) concentrations in the hemolymph and different tissues of P. clarkii. Two hundred crayfish were collected and were divided into two groups; field crayfish (FCF) that were dissected directly and the depurated crayfish (DCF) that were housed for 21 days in glass tanks. Procambarus clarkii were dissected to determine the total hemocytes count and heavy metal concentrations in the hemolymph, abdominal muscles, and hepato-pancreas. The results showed that the depuration has led to a significant decrease in the total count of hemocytes, however, it increased the percentages of semi-granular cells, granulocytes and decreased the percentage of hyalinocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed different protein patterns between FCF and DCF. Not all the selected heavy metals were detected in the collected water samples and in the investigated tissues. Depuration has led to a significant decrease of the heavy metals, if present, in the hemolymph, abdominal muscles, and hepato-pancreas. Collectively, the depuration resulted in significant decreases in the total hemocytes count and eliminated the selected heavy metals from the different tissues of P. clarkii.

Molecular markers for hemocyte subpopulations in crayfish Cherax quadricarinatus

Semigranular cells (SGCs) and granular cells (GCs) are two dominant groups of circulating hemocytes in crayfish Cherax quadricarinatus. Molecular markers are required for the clear classification of the hemocytes and the research of their function and differentiation. In this study, we compared the protein content of GCs and SGCs by using two workflows: one-dimensional gel electrophoresis followed by LC-MS/MS and in-solution digestion of cell lysate followed by LC-MS/MS. Cell type-specific proteins were identified, and their expression in SGCs and GCs was further investigated by RT-PCR, Western blotting, and immunofluorescence analysis. Three molecular markers for GCs (peroxinectin, a mannose-binding protein, and prophenoloxidase-activating enzyme 2a) and three molecular markers for SGCs (a vitelline membrane outer layer protein I-like protein, a C-type lectin, and a peptidase) were identified. The application of some of the markers in Eriocheir sinensis was also analyzed. These molecular markers are useful tools for the research of crustaceans hemocytes.

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

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

In vivo study of a novel protein kinase C that mediates immunocompetence and catecholamine biosynthesis in hemocytes of Litopenaeus vannamei by using its potential competitive inhibitor, bisindolylmaleimide I

This study applied bisindolylmaleimide I (BSM), a pharmacological competitive inhibitor of protein kinase C (PKC) enzymatic activity, at 1.25 pmol shrimp−1 for 60 min to investigate the potential involvement of PKC in signal transduction pathways in the hemocytes of Litopenaeus vannamei. A novel PKC in L. vannamei (LvnPKC) was identified and characterized and was determined to be involved in mediating the neuroendocrine-immune regulatory network. The hemocytes of L. vannamei that receive BSM exhibit significantly decreased PKC activity and LvnPKC gene and protein expression levels. Furthermore, the total hemocyte count, hyaline cells, and semigranular cells increased significantly along with significant decreases in granular cells, and meanwhile, the significantly increased phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and neutrophil extracellular trap were observed; however, phagocytic activity decreased significantly. In a molecular model, the gene expressions of lipopolysaccharide- and β-1,3-glucan-binding protein, peroxinectin, cytosolic manganese SOD, mitochondrial manganese SOD, and copper/zinc SOD in the hemocytes of L. vannamei that had received BSM decreased significantly, but prophenoloxidase I increased significantly. In catecholamine biosynthesis, tyrosine, dopamine, and norepinephrine decreased significantly in the hemocytes of L. vannamei that had received BSM, and l-dihydroxyphenylalanine increased. Moreover, tyrosine hydroxylase (TH) activity increased significantly, whereas TH and dihydroxyphenylalanine decarboxylase gene expression decreased significantly. These findings suggest that BSM inhibits PKC activity in hemocytes in which LvnPKC gene and protein expression are also inhibited. Additionally, the hemocytes’ immunocompetence, including their prophenoloxidase and antioxidant systems, phagocytic activity, and catecholamine biosynthesis, was disrupted, confirming the roles of LvnPKC in mediating the neuroendocrine-immune regulatory network in hemocytes.

Host-parasite interactions: A study on the pathogenicity of Mesanophrys sp. and hemocytes-mediated parasitic resistance of swimming crabs (Portunus trituberculatus) at different temperatures

Mesanophrys sp. is a ciliated parasite that invades the hemolymph of swimming crabs (Portunus trituberculatus) and reproduces rapidly under low temperatures. We conducted an in vitro and in vivo interaction assays between the P. trituberculatus hemocytes and the Mesanophrys sp. at different temperatures to understand the pathogenicity of Mesanophrys sp. and the antiparasitic responses of crab hemocytes.The results showed that infecting crabs with the Mesanophrys sp. caused a time-dependent decrease in the number of crab hemocytes (including total, granular, semi-granular, and hyaline cells) compared with the two control groups (injecting seawater-treatment and no-Mesanophrys sp. treatment groups).The in vitro interaction between hemocytes and the Mesanophrys sp. resulted in the lysis of the crab hemocytes to varying degrees. The survival rate of the hemocytes decreased with time in both Mesanophrys sp. and non-Mesanophrys sp. treatment (control) groups across all temperatures (24 °C, 20 °C, 16 °C, and 12 °C). However, the survival rate of hemocytes in the Mesanophrys sp. treatment groups (24 °C: 5.3 ± 1.2, 20 °C: 6.0 ± 1.4, 16 °C: 5.7 ± 1.7, and 12 °C: 5.67 ± 0.47) showed a faster and greater declining trend compared to the non- Mesanophrys sp. groups (24 °C: 32.7 ± 2.5, 20 °C: 32.3 ± 1.2, 16 °C: 31.3 ± 1.70, and 12 °C: 36.0 ± 1.63). Additionally, a decrease in temperature (from 24 °C to 12 °C) greatly decreased the cell integrity of hemocytes mixed with the culture medium of Mesanophrys sp. than the groups with culture medium without Mesanophrys sp.The population density of Mesanophrys sp. was significantly higher in the control group (A: with no-hemocytes or no-hemocytes culture medium) than the treatment groups (B: with hemocytes culture medium or C: with hemocytes) at 6–18 h. However, the population densities of Mesanophrys sp. in treatment groups B (13,444 ± 874 ind/mL) and C (52,178 ± 1138 ind/mL) were significantly higher than the control group (A) (1567 ± 258 ind/mL) after 18 h. Conversely, the population density, natural population growth rate, generation time, and length to width ratio of Mesanophrys sp. incubated with hemocytes increased significantly with decreasing temperature than the non-hemocytes group.In summary, the Mesanophrys sp. infects and lyses the hemocytes of the swimming crab and consumes the lysate for its proliferation. Decreasing the culture temperature from 24 °C to 12 °C reduced the resistance of crab hemocytes to the parasite Mesanophrys sp. but enhanced the impact of the Mesanophrys sp. on hemocytes.

Identification and function analysis of an immune deficiency homolog in swimming crab, Portunus trituberculatus

The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt-IMD) was identified from a marine crab, Portunus trituberculatus. The Pt-IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt-IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt-IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt-IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt-IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt-IMD expression was also knock-down by RNAi to determine the function of Pt-IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus, after Pt-IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells.