Differential Hemocyte Counts Research Articles

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.

Expression of octopamine/tyramine receptors and immune regulation in Litopenaeus vannamei under acute and chronic thermal stress

This study investigated the impact of hyperthermal (34 °C) and hypothermal (14 °C) stress on the expression of the octopamine/tyramine receptor (LvOA/TA-R) and immune parameters in Litopenaeus vannamei, which is a species critical to the aquaculture industry. Given the sensitivity of aquatic organisms to climate change, understanding the physiological and immune responses of L. vannamei to temperature variations is essential for developing strategies to mitigate adverse effects. This research focuses on the immune response and expression changes of LvOA/TA-R under acute (0.5, 1, and 2 h) and chronic (24, 72, and 168 h) thermal stress conditions. Our findings reveal that thermal stress induces changes in LvOA/TA-R expression and impacts immune responses. Immune parameters such as total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, lysozyme activity, clearance efficiency, and phagocytosis exhibited a general trend of significant decline under the stress conditions. LvOA/TA-R had a higher expression in haemocyte under hyperthermal stress. The study elucidated that thermal stress modifies the expression of the LvOA/TA-R and diminishes immune functionality in L. vannamei, underscoring the potential influence of climate change on industry.

Response of the terrestrial isopod Porcellio scaber to lipopolysaccharide challenge after microplastic and insecticide exposure

The exposure of organisms to microplastics could compromise their ability to cope with other environmental stressors, such as infections. In this context, we investigated the effects of a 14-day exposure of the terrestrial isopod Porcellio scaber to tire particles in soil (1.5 % w w−1 dry weight) on the organisms' response to a secondary exposure, i.e., injection of the bacterial endotoxin lipopolysaccharide. In addition, the insecticide chlorpyrifos (2 mg kg−1 dry weight) was tested as a positive control. The survival and immune response of P. scaber was assessed at the end of the 7- and 14-day primary exposure and two days after the secondary exposure, by analyzing selected haemolymph immune parameters (total haemocyte count, differential haemocyte count, and haemocyte viability). No change in survival was observed after primary exposure of P. scaber to tire particles or chlorpyrifos. However, primary exposure to chlorpyrifos triggered a strong activation of the immune response, which was not the case following exposure to the tire particles. Further injection of lipopolysaccharide into the body did not affect the survival of animals exposed to tire particles or chlorpyrifos, while a strong immunomodulatory change was observed, particularly with chlorpyrifos, and to some extent, tire particles. Based on these results, we conclude that exposure of P. scaber to tire particles or chlorpyrifos has no significant effect on the susceptibility of the organism to lipopolysaccharide in terms of their mortality, but primary exposure to an insecticide significantly modulates the immune response of the organisms to a second stressor. We discuss the “stress on stress” approach for testing low-toxic substances, such as microplastics, where an environmentally realistic exposure is followed by a secondary exposure.

Responses of continuous cadmium exposure on some biological and physiological performance of cotton bollworm Helicoverpa armigera

AbstractA detailed effect of continuous exposure of Cd on an important pest of crops like Helicoverpa armigera is lacking in the current literature. The cadmium was provisioned at 1.25, 2.5 and 5 mg/kg in the artificial diet and fed to newly hatched larvae for at least three subsequent generations. The total developmental period and fecundity were severely affected with no larval hatching in treated insects. Total haemocyte count and differential haemocyte count (i.e., granulocytes and plasmatocytes) were decreased significantly compared to controls in treated insects. The antioxidant system (i.e., catalase, glucose‐6‐phosphate dehydrogenase, superoxide dismutase and peroxidase), and the two detoxifying agents glutathione‐s‐transferase showed increased activity compared to controls. The results of the acetylcholinesterase assay showed a decrease in activity. Increased rate of aspartate aminotransferase, alanine aminotransferase, uric acid and decreased rate of alkaline phosphatase and lactate dehydrogenase in the first generation but showing an increasing trend in next two generations. The acid phosphatase activity was increased in the first two generations but significantly reduced in the third generation. The discrepancy in all physiological activities corresponded with the histological study of midgut infrastructure. It is inferred from the obtained results that cadmium continuous exposure affects every system of any organism that may perform irreparable damages.

Effect of supersaturated dissolved oxygen on growth-, survival-, and immune-related gene expression of Pacific white shrimp (Litopenaeus vannamei).

Oxygen concentration is an essential water quality parameter for aquaculture systems. Recently, supersaturated dissolved oxygen (DO) has been widely used in aquaculture systems to prevent oxygen depletion; however, the long-term effects of supersaturated DO exposure on aquatic animals have not been studied. In this study, we examined the effects of supersaturated DO on the growth, survival, and gene expression of Pacific white shrimp (Litopenaeus vannamei). Specific pathogen-free shrimp with a body weight of 8.22 ± 0.03 g were randomly assigned to two groups with four replicates at a density of 15 shrimps per tank. Shrimp were cultivated in recirculating tanks containing 50 L of 15 ppt seawater in each replicate. Oxygen was supplied at 5 mg/L to the control tanks using an air microbubble generator and at 15 mg/L to the treatment tanks using a pure oxygen microbubble generator. Shrimp were fed commercial feed pellets containing 39% protein at 4% of their body weight per day for 30 days. Average daily growth (ADG) and feed conversion ratio (FCR) were determined on days 15 and 30. Shrimp molting was measured every day. Individual hemolymph samples were obtained and analyzed for total hemocyte count, differential hemocyte count, and expression of growth- and immune-related genes at the end of the experiment. Long-term exposure to supersaturated DO significantly affected shrimp growth. After 30 days of supersaturated DO treatment, the final weight and ADG were 14.73 ± 0.16 g and 0.22 ± 0.04, respectively. Shrimp treated with normal aeration showed significantly lower weight (12.13 ± 0.13 g) and ADG (0.13 ± 0.00) compared with the control group. FCR was 1.55 ± 0.04 in the treatment group and 2.51 ± 0.09 in the control group. Notably, the shrimp molting count was 1.55-fold higher in the supersaturated DO treatment than in the supersaturated DO treatment. The expression of growth-related genes, such as alpha-amylase, cathepsin L, and chitotriosidase, was 1.40-, 1.48-, and 1.35-fold higher, respectively, after supersaturated DO treatment. Moreover, the treatment increased the expression of anti-lipopolysaccharide factor, crustin, penaeidin3, and heat shock protein 70 genes by 1.23-, 2.07-, 4.20-, and 679.04-fold, respectively, compared to the controls. Supersaturated DO increased growth and ADG production and decreased FCR. Furthermore, enhanced immune-related gene expression by supersaturated DO may improve shrimp health and reduce disease risk during cultivation.

Evaluating the effect of temperature on Penaeus vannamei culture in a heterotrophic system and experimentally infected with the infectious myonecrosis virus

Causing economic losses in Brazil and Indonesia between 2002 and 2011 of over US$ 1 billion, infectious myonecrosis (IMN) constitutes a disease of mandatory notification to WHO (World Health Organization) and is responsible for constant damages in the production of the marine shrimp Penaeus vannamei. Although environmental factors such as high thermal amplitude, high concentration of nitrogen compounds and abrupt changes in salinity can alter the infection rate of this disease, there are still no studies. Thus, the present work aimed to evaluate the influence of temperature on the infectivity of IMNV in P. vannamei culture in a heterotrophic system. The experiment lasted 42 days, divided into two stages: (i) subjecting the shrimps to different temperatures (26 °C, 29 °C, 32 °C, and 35 °C) for 21 days; and (ii) viral challenge with IMNV via intramuscular injection (100 μL) also for 21 days, with evaluation of the shrimps by nested-PCR, histopathology and total and differential hemocyte count. The results obtained indicated that shrimps submitted to the highest temperatures evaluated (32 °C and 35 °C) had the highest post-viral challenge survival rates (85.42% and 93.75%, respectively), with less presence of lesions suggestive of IMN, although these shrimps were still positive by nested-PCR. No significant differences were observed in total hemocyte counts between pre- and post-challenge treatments (P > 0.05). In conclusion, the use of a temperature of 35 °C reduced mortality and viral load but altered the feed absorption, generating a reduction in zootechnical performance.

The Effect of Hyperthermia on the Immune Response of Vannamei Shrimp Infected with IMNV

The purpose of this study was to determine the effect of hyperthermia on the immune response of shrimp infected by IMNV. Vannamei shrimp were challenged with IMNV and reared at temperatures 30ºC (Sk30), 31ºC (S31), 32ºC (S32) and 33ºC (S33) repectively. The temperature of shrimp without IMNV infection was 300C as control. During the study, shrimp immune response measurements were Total Hemocyte Count (THC), Differential Hemocyte Count (DHC), Respiratory Burst (RB) activity and Phenoloksidase (PO) activity. Generally, the results showed that the response of shrimp decreased along with hyperthermic condition. Shrimp infected with IMNV, decreased immune response especially at temperatures of 32ºC and 33ºC.

Growth inhibitory, immunosuppressive, cytotoxic, and genotoxic effects of γ-terpinene on Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae)

γ-Terpinene, a monoterpene widely present in essential oils of many medicinal and aromatic plants with numerous biological properties, was evaluated for its insecticidal activity against melon fruit fly, Zeugodacus cucurbitae (Coquillett). Different concentrations (5, 25, 125, 625, and 3125 ppm) of γ-terpinene along with control were fed to larvae of melon fly. The number of pupae formed and adults emerged declined significantly after treatment. Morphologically deformed adults and pupae were also observed. The developmental duration too prolonged in treated larvae. Food assimilated, mean relative growth rate, larval weight gain, and pupal weight also declined. In the larvae treated with LC30 and LC50 concentrations, there was a decline in the titers of phenoloxidase and total hemocyte count, and variations were observed in the differential hemocyte count, suggesting an immunosuppressive effect of γ-terpinene on melon fly. Both concentrations also led to an increase in the apoptotic and necrotic cells as well as decrease in the viable hemocytes in the circulating hemolymph of treated larvae. Comet parameters (tail length, % tail DNA, tail moment, and olive tail moment) of γ-terpinene fed larvae increased significantly. Given the observed effects of γ-terpinene on normal developmental and nutritional physiology, its immunosuppressive properties, and its potential for genome damage, it can be considered for incorporation into integrated pest management strategies for controlling Z. cucurbitae.

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.

Levamisole Influences the Immune Response of Freshwater Prawn, <i>Macrobrachium rosenbergii</i> and its Resistance to Nitrite Stress and <i>Aeromonas hydrophila</i> Infection

The present study evaluated the effectiveness of dietary levamisole in the modulation of innate immunity and reducing the percent mortality against nitrite stress or Aeromonas hydrophila infection in giant freshwater prawn, Macrobrachium rosenbergii. Haemolymph agglutinin and total protein levels, lysozyme activity, phenoloxidase (PO) activity, total and differential haemocyte counts, induced nitrite stress and mortality (%) against Aeromonas hydrophila challenge were measured in sub-adult M. rosenbergii fed with diets containing levamisole at 0, 125, 250 and 500 mg/kg feed for 7 or 14 days. M. rosenbergii fed with a diet containing 250 mg levamisole/kg feed for 14 days showed significant (P<0.05) increase in haemagglutination titre, PO activity, undifferentiated haemocyte count, resistance to nitrite stress and survival against A. hydrophila challenge. On the contrary, graded levels of levamisole feeding for 7 days failed to modulate most of the immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of levamisole in the diet at 250 mg/kg feed for 14 days in sub-adult M. rosenbergii could enhance the immune ability and increase its resistance to A. hydrophila infection and nitrite stress.

Dietary administration of mangosteen, Garcinia mangostana, peel extract enhances the growth, and physiological and immunoendocrinological regulation of prawn, Macrobrachiumrosenbergii.

In this study, we investigated the potential immunostimulatory effects of mangosteen (Garcinia mangostana) peel extract on Macrobrachium rosenbergii, specifically in enhancing immunity and resistance against Lactococcus garvieae. We employed a dietary administration approach to assess the impact of different extract preparations from mangosteen peel, namely mangosteen peel powder (MPP), boiled mangosteen peel powder (MPB), and mangosteen peel extract (MPE). Following the administration of mangosteen peel extract, we evaluated growth performance, innate immune parameters, and disease resistance in the prawns. The results revealed a significant increase in total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), as well as phagocytic activity and clearance efficiency against L. garvieae. Based on these findings, we suggest that mangosteen peel extract can be utilized as an immunostimulant for prawns through dietary administration, regulating immune responses and enhancing resistance against pathogens by modulating carbohydrate metabolism.

Survival and Immune Responses of F1 Transgenic Tiger Shrimp Penaeus monodon Against White Spot Syndrome Virus (WSSV)

Transgenic technology has been applied to tiger shrimp Peneus monodon to produce a resistant strain to white spot syndrome virus (WSSV) by antiviral gene overexpression. The founders (F0) transgenic tiger shrimp have been successfully bred to produce the first generation (F1). The present study aimed to evaluate the survival and immune responses of F1 transgenic tiger shrimp against WSSV. The F1 transgenic and non-transgenic shrimp (control) with an average weight of 7.93±1.49 g were collected from the controlled brackish water ponds and stocked in a 20 L fiberglass tank with a density of 5 shrimp/tank. The transgenic shrimps were confirmed by a PCR assay. The shrimp were intramuscularly injected with WSSV to evaluate the performance between transgenic and non-transgenic. The survival (SR) was observed daily after the challenge test. Measurement of immune responses, namely total haemocyte count (THC), differential haemocyte count (DHC), prophenoloxidase (proPO) activity, and RNA content in haemolymph was conducted on before challenge, and on the 1st, 3rd, and 5th days after challenge. The survival and immune responses were statistically analysed by the t-student test. The results showed that the SR of transgenic (52.0%), with a relative percentage survival of 47.82%, was greater (P<0.05) than the control shrimp (8.0%). The THC, proPO, and RNA content of the transgenic shrimp was higher (P<0.05) than the non-transgenic shrimp. The results suggested that the transgenic tiger shrimp, due to antiviral gene overexpression, increased the resistance against WSSV.

Biochemical and histopathological responses of Biomphalaria alexandrina to RIPEX (plant growth regulator)

BackgroundPlant growth regulators are widely used in agriculture for increasing the growth and ripening of plants, but they endanger the aquatic ecosystem. The current study assessed the effect of sublethal exposure to RIPEX 48% EC concentrations (8 and 16 µL/L) on oxidative stress parameters, sex hormones, immune potential enzymes, differential hemocyte counts, and the histopathology of digestive glands and ovotestis in Biomphalaria alexandrina snails.ResultsRIPEX exposure caused an overall increase in Superoxide dismutase and Glutathione-S-Transferase activities in B. alexandrina. However, extreme RIPEX exposure inhibits SOD activity in snails. Malondialdehyde activity showed an increase in B. alexandrina exposed to both concentrations after all exposure periods. RIPEX also caused a significant increase in testosterone in snails exposed to 16 µL/L, it did, however, reduce hormone levels in snails exposed to 8 µL/L at 7 days. Regarding estradiol, there was a significant increase after 3 days of exposure to 16 µl/L and 7 days of exposure to 8 µL/L. RIPEX exposure also increased the activities of Myeloperoxidase and Adenosine deaminase enzymes in the digestive glands of snails. It increased the total hemocyte count of exposed snails as well as the number of granulocytes. Snail digestive glands and ovotestis showed pathological alterations after 7 days of RIPEX exposure.ConclusionsThese findings suggest that RIPEX is toxic to B. alexandrina and that this snail can be used as a bioindicator for environmental contamination with plant growth regulators.

Beta-cyfluthrin-Induced Alterations in the Total and Differential Haemocytes Count in the Red Cotton Bug, Dysdercus koenigii (Fabricius,1775)

Dysdercus koenigii is a major global pest of cotton that causes severe economic loss. Among several control measures, pyrethroids are frequently used toxicants because of high efficacy at low dosages and relative safety. Since haemocytes are biomarkers of the physiological response and immunity of insect which determine the insecticide efficacy, the current study assessed the effect of a pyrethroid, β-cyfluthrin, on the total and differential haemocyte counts of D. koenigii. Haemolymph was collected from the fifth instars after the topical application of β-cyfluthrin (0.8, 1.6, 3.2, 6.4 and 12.8 mg/L) on the thoracic tergum. The haemolymph of control nymphs revealed 5270 haemocytes/mm3 which decreased instantly by 1.4-3.1-fold on β-cyfluthrin exposure; more reduction observed at lower dosages. Increase in exposure duration and β-cyfluthrin dosages fluctuated the count considerably, eventually raising them at lower dosages and diminishing at higher dosages. Among five kinds of haemocytes recorded in the haemolymph, the β-cyfluthrin exposure increased %prohaemocytes count; diminished %granulocytes and %plasmatocytes count while spherulocyte and oenocyte counts were inconsistent. The alterations in haemocyte counts indicate the immunity response trigger in D. koenigii due to β-cyfluthrin-induced stress. Further investigations may decipher the mechanisms involved and help to formulate the strategies for its management in fields.

Effects of parasitism by the braconid wasp, Bracon hebetor (Hymenoptera: Braconidae), on the host hemolymph and phenoloxidase activation of the rice moth, Corcyra cephalonica larvae (Lepidoptera: Pyralidae)

BackgroundBiological control of insect pests is an economic, eco-friendly and harmless approach to integrated pest management strategies. Bracon hebetor (Say) (Hymenoptera: Braconidae) considers a polyphagous ectoparasitoid of various pests of the order Lepidoptera. The parasitized host insects' defense mechanisms are triggered as a result of the parasitic wasps' injury and penetration. Thus, induce the host cellular and humoral immune responses through a blend of secretions injected into the host body. The present study was designed to evaluate the efficacy of B. hebetor on the immune response of the parasitized full grown larval instar of the rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) under natural envenomation.ResultsOn cellular level, the ultrastructure examination of the hemocytes displayed a considerable structural deformation in hemocyte morphology of the detected types of the hemocytes. Moreover, the effects of parasitism on both differential hemocyte counts (DHCs) and total hemocyte counts (THCs) were investigated. The number of prohemocytes (PR) (40.33 ± 5.61, 43 ± 8.33 and 26 ± 2.31) was statistically differed after (24, 48 and 72 h) of parasitism, respectively, compared to unparasitized (31.33 ± 6.49) larvae. Similar observations were recorded in plasmatocytes (PL) before and after the parasitism. However, Spherulocytes (SP) and Oenocytoids (OE) were recorded in the hemolymph with little abundance. On the other side, the quantitative analysis of total hemolymph proteins (THP) provoked a significant effect of considering parasitized and non-parasitized larvae. As the Phenoloxidase (PO) cascade plays a critical role in immune defenses, so the substantial activation of PO in the host's hemolymph following successive hours of parasitism compared to unparasitized larvae using L-DOPA as a substrate indicated the induction of larval immune system. The parasitized larvae showed a gradual increase in the PO activity (0.442 ± 0.103) after 24 h reached up to (1.482 ± 0.272) at the end of parasitism (72 h) in comparable to unparasitized larvae (0.177 ± 0.0.044).ConclusionThe present investigations clarified the efficacy of B. hebetor parasitism on the host immune mechanism, which will enable the progress of sustainable stored product protection approaches for the control of an important pest rice moth C. cephalonica.

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.

Effectiveness of various cacao pod husk extraction byproducts in promoting growth and immunocompetence in Litopenaeus vannamei

Extracts from plant products can promote growth, can act as immunostimulants, and have antibacterial and antiparasitic properties. These extracts can be used as alternatives to the chemical treatments commonly used to prevent and control disease in aquatic species. Research on the subject has focused on identifying invasive plants or agricultural waste products that can be used as immunostimulants. The present study further identified an optimal means of extracting pectin from cacao pod husks to promote growth performance and immunocompetence in Litopenaeus vannamei that would both reduce production costs and enable waste recycling. The byproducts of pectin extraction from cacao pod husks, that is, dried cacao pod husk powder (DCP), steamed DCP (sDCP), hot water–treated cacao pod husk powder (HCP), hot water–treated cacao pod husk supernatant (HCS), and cacao pod husk pectin (CPH pectin), were used to create five experimental diets, which were administered to five groups. The control group was fed a basal diet. The growth and immunocompetence of the shrimp were determined after 30, 60, 90 and 120 days of feeding. To identify the most cost-effective means of obtaining dried cacao pod husks, this study firstly determined the costs and effectiveness of the sun-drying, dehumidification, and heated-wind drying techniques. According to the results of growth performance, the CPH pectin group had higher survival but lower weight gain than the DCP, sDCP, HCP, and HCS groups did. At 30, 60, and 90 days, the clearance efficiency of the experimental groups was higher than that of the control group. At 60 days, the experimental groups had significantly higher phagocytic activity than the control group did. However, at 30 and 90 days the HCP, HCS and CPH pectin group had higher phagocytic activity. The total hemocyte count, differential hemocyte count, phenoloxidase activity, and respiratory bursts of the CPH pectin group were higher at 30 days but the same as those of the control group at 60 and 90 days. After 120 days of feeding trial, the resistance of L. vannamei fed with diets containing byproducts of pectin extraction from cacao pod husks significantly enhanced compared to that in BD group when they were infected with Vibrio aliginolyticus for 168 h, and the related higher survival rate can be observed in HCP, HCS and CPH pectin groups. The study findings suggest that diet-administered HCP and HCS have long-term immunostimulant potential and that CPH pectin has potential in the early stages of feeding. In addition, when heated air drying was employed, a moisture level of below 10% was obtained within 12 h. The results of this study indicate that adding HCP obtained from heated air–dried cacao pod husks to the feed of L. vannamei is the most cost-effective and sustainable means of promoting long-term growth performance and immunocompetence in the species.

Plant species with higher chemical defences enhance herbivore cellular immunity with differential effectiveness against two parasitoid species

Abstract Insect herbivores simultaneously experience bottom‐up effects of plant defensive chemistry and the top‐down effects of natural enemies. At the intersection of these effects are herbivore immune systems, herbivore traits that have largely been overlooked in studies of plant‐insect interactions. Most previous studies have demonstrated compromised immunity of herbivores that feed on plants with higher defensive chemistry. Many studies have used embedded microfilaments or silica beads as proxies for parasitoid eggs. Yet, parasitoids may evade or suppress host immune responses by injecting venom and calyx fluid, or through modifications of their egg surface structure, necessitating studies that include all three trophic levels to obtain a complete picture of how plant traits may modulate herbivore immunity. Here, we examined the effect of host plant species that differ in glucosinolate (anti‐herbivore compounds produced by plants in the Brassicaceae) concentrations on the immune status of an herbivore and its consequences for two species of parasitoids with different life history traits. We found that larvae of the butterfly Pieris rapae that fed on field mustard Brassica rapa, which contain 52‐fold higher glucosinolate concentrations than collards B. oleracea, attained lower body weights and experienced prolonged development to adulthood. Yet, caterpillars that fed on B. rapa had enhanced cellular immunity, as measured by total and differential haemocyte counts as well as melanization capacity, compared to larvae that fed on B. oleracea. In turn, the likelihood that at least some eggs in clutches of the gregarious endoparasitoid Cotesia glomerata would be encapsulated, leading to a reduction in brood size, were three times greater when their host caterpillars fed on B. rapa compared to B. oleracea. Interestingly, eggs of the solitary endoparasitoid Cotesia rubecula were rarely encapsulated irrespective of the host plant on which their host caterpillar fed. Therefore, our results suggest that plant defence metabolites can influence the expression of herbivore immunity, but the effectiveness of this response strongly depends on the identity of the parasitoid and its ability to evade the caterpillar immune response, and possibly the evolution of these trophic interactions in non‐native systems. Read the free Plain Language Summary for this article on the Journal blog.

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<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%

The immunostimulatory effects of Theobroma cacao L. pod husk extract via injection and dietary administrations on Macrobrachium rosenbergii and its resistance against Lactococcus garvieae

The extract from Theobroma cacao L, pod husk served as the immunostimulant to enhance the immunity and resistance against Lactococcus garvieae of Macrobrachium rosenbergii. In this study, we employed the injection method and dietary administration method to determine the effect of cocoa pod husk (CPH) on M. rosenbergii. The non-specific immune parameters and disease resistance were evaluated after the prawn injected with 1 μg prawn−1 CPH extract (C1), 3 μg prawn−1 CPH extract (C3), and 5 μg prawn−1 CPH extract (C5) for 1, 3, and 7 days. The results showed a significant increase of total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity and clearance efficiency to L. garvieae. The non-specific immune parameters, physiological parameters, and disease resistance and growth performance were evaluated after the prawn fed with 1 g kg−1 CPH extract diet (CD1), 3 g kg−1 CPH extract diet (CD3) and 5 g kg−1 CPH extract diet (CD5). The results showed a significant increase in all immune parameters and showed a significant decrease in physiological parameters. No significant difference was observed in growth performance of prawn fed with the CPH containing diet. Both injection and dietary method showed a significant increase in disease resistance against to L. garvieae. We therefore recommend that CPH extract can be used as a immunostimulant for prawn by dietary administration to regulate immune responses, and carbohydrate metabolism lead to enhance resistance against pathogen.