Differential Hemocyte Counts Research Articles

Methanolic Extract of Winter Cherry Causes Morpho-Histological and Immunological Ailments in Mulberry Pyralid Glyphodes pyloalis.

The effect of Withania somnifera a medicinal plant seed extract was tested against lesser mulberry pyralid, a potential pest of mulberry. The mulberry leaves were used for silk production in rural areas of northern Iran. The extract was administered orally by leaf dipping method in two lower (5%W/V) and higher (15%W/V) dosages to third instar larvae (<24 h) for biological assays and to fifth instar larvae (<24 h) for Physiological studies. The results showed formation of larvoids (Ls), larval-pupal intermediates (LPIs), pupoids (Ps) and pupal-adult intermediates (PAIs). The results showed increased larval duration by 1.7 and 2 folds in 5 and 15% treatment, respectively. Fecundity of resultant adults was decreased by 1.2 and 1.3 in 5 and 15% treatment, respectively. Except approximate digestibility (AD) and consumption index (CI) all other feeding indices showed reduction. The feeding deterrence was prominent at 15% (87%) and 5% showing 48% deterrence. Our enzymatic and non-enzymatic assessments upon treatment showed reduction in key components, except detoxifying enzymes. However, the activity of an important enzyme involved in cuticle hardening and immunity called phenoloxidase was reduced. We also investigated the histology of midgut for further analysis and found drastic changes in main cellular elements. Immunological changes following treatment was noticeable in reduced Total Hemocyte Count but surprisingly increased Differential Hemocyte Count. However, the hemocytes structure was extremely damaged. The reduced number of eggs in treated but survived adults indicated reduced ovaries, with vacuolization both in trophocytes and oocytes. The key chemical compounds showed reductions particularly at 15%. The present results are concomitant with few earlier studies on this medicinal plant and deserve further studies particularly in deriving key chemicals that alter metamorphosis similar to insect growth regulators.

The neonicotinoid insecticide imidacloprid, but not salinity, impacts the immune system of Sydney rock oyster, Saccostrea glomerata

The broad utilisation of neonicotinoids, particularly imidacloprid (IMI), in agriculture has led to unplanned contamination of aquatic systems around the world. The sublethal effects of individual pesticides on the immune system of oysters, as well as their combined effects with other environmental stressors that fluctuate in estuarine environments, such as salinity, are yet to be investigated in ecotoxicology. We investigated the acute (4 d) toxicity of IMI in two salinity regimes on the immune parameters of Sydney rock oysters (SRO), including total hemocyte counts (THC), differential hemocyte counts (DHC), phagocytosis and hemocyte aggregation (HA), hemolymph protein expression and enzyme (catalase (CAT), glutathione S-transferase (GST) and acetylcholinesterase (AChE)) activities. Environmentally relevant concentrations of IMI were found to cause an increase in THC, induce GST activity, reduce HA, and inhibit AChE activity. However, DHC, CAT activity and phagocytosis were not significantly impacted at any test concentration at either salinity. IMI concentrations ≥0.01 mg/L significantly altered the expression of 28 proteins in the hemolymph of SRO, including an increase in the relative expression of extracellular superoxide dismutase, severin, ATP synthase subunit beta, as well as stress response proteins (heat shock proteins, serine/threonine-protein kinase DCLK3 and peroxiredoxin-1), and a decrease/absence of collagen alpha-4 (VI) and alpha-6 (VI) chain, metalloendopeptidase, L-ascorbate oxidase, transporter, CEP209_CC5 domain-containing protein and actin. This study indicates that the immune system of SRO can be impacted at environmentally relevant concentrations of IMI, but reduced salinity does not appear to influence the toxicity of this insecticide.

Susceptibility of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) to entomopathogenic nematodes with regard to its immune response

The red palm weevil (RPW) Rhynchophorus ferrugineus Olivier is the most devastating pest of date palm, with a severe impact on cultivation and production. The virulence of five species/strains of entomopathogenic nematodes (EPNs), Steinernema scapterisci (Ss), Steinernema sp. (SII), Steinernema abbasi (Ab), Steinernema glaseri (Sg), and Heterorhabditis bacteriophora (HP88) was tested against larval and adult stages of RPW under semi-field laboratory conditions. Total and differential haemocyte counts were determined, and protein analysis and histopathological study of larvae injected with SII and HP88 were performed. Susceptibility results revealed that the larval stage was much more susceptible to Sg and HP88 than the adult stage. Larval mortality rate was as high as 100% and 92%, respectively, with concentrations of 1250infective juveniles (IJs)/pot, while adult mortality reached 48% and 60%, respectively, with concentrations of 20000IJs/pot at 9 d post infection with Sg and HP88. In general, SS, SII, and Ab appeared to be more virulent during the adult stage than the larval stage. They recorded mortality rates of 88%, 88%, and 72%, respectively, for the larval stage, and 100%, 100%, and 84%, respectively, for the adult stage at concentrations of 20000IJs/pot at 9 d post infection. A severe decline in the total and differential haemocyte counts (THCs and DHCs) at 12 h post-injection with SII and HP88 at 20IJs/larvae was observed. Similarly, the effect on total haemolymph protein of the normal and infected larvae was reflected by the appearance and disappearance of protein bands in addition to changes in the protein band concentration. Notably, remarkable changes in the cuticle, in the form of disruption to the outer epicuticle, splitting of the epidermal layer, and disorganization of the musculature region, were observed. In addition, notable histological effects were seen in the midgut region at 12 h post injection with 20IJs/larvae of HP88 and SII. Our results demonstrate that R. ferrugineus is unable to defend itself against EPNs. These results indicate an effective and natural biological control strategy with potential for application as an eco-friendly and inexpensive insecticide against the adult and larval stages of the RPW.

The aggregation pheromone phenylacetonitrile: Joint action with the entomopathogenic fungus Metarhizium anisopliae var. acridum and physiological and transcriptomic effects on Schistocerca gregaria nymphs

The combined use of entomopathogenic fungi and sublethal rate of chemical insecticides or other biological control agents have been proposed as an environmentally and sustainable strategy in the control of locust pests. In this paper, the quarter and the half of the recommended dose of Metarhizium anisopliae var. acridum (¼ and ½ Ma) and the aggregation pheromone (Phenylacetonitrile: PAN) were applied simultaneously and sequentially to Schistocerca gregaria fifth-instar nymphs. In addition, the physiological effects of PAN on locusts were assessed at the behavior, immune response, and biochemical level by evaluating for glutathione-S-transferase (GST), acetylcholinesterase inhibition (AChE), and malondialdehyde accumulation (MDA). Results showed that simultaneous application of PAN and the entomopathogenic fungus exhibited additive interaction. Synergistic interaction was also demonstrated when nymphs were exposed to PAN first, then treated with M. anisopliae var. acridum. Behavioral bioassay revealed that fifth-instar nymphs avoided the PAN odour and tended to remain away from the stimulus cup. In the choice assay, the pheromone significantly repelled the locusts at 2, 4, and 6 h of exposure which selected the PAN-free arena chamber. Moreover, treated nymphs become hyperactive and disoriented as evidenced by the cumulative distance travelled and the trajectory of locusts during the experiment. Immunological studies showed that PAN significantly decreased the differential haemocyte counts (prohemocytes and plasmatocytes) with a dose-response relationship. Data of biochemical analyzes showed that the PAN exposure reduced the activity of acetylcholinesterase and induced significantly the glutathione S-transferases and MDA concentration in the desert locust fifth-instar nymphs. Moreover, transcriptomic responses to the PAN exposure were evaluated using gene expression levels of CYP540 and GST. The transcript levels showed an up-regulation in GST expression level particularly in nymphs exposed for 4 and 6 h. A significant increase in CYP450 transcript level was also observed after 2 h of exposure, which decreased significantly after 4 and 6 h.

Environmental concentrations of sulfamethoxazole increase crayfish Pacifastacus leniusculus susceptibility to White Spot Syndrome Virus

Antibiotics used for humans and livestock are emerging as pollutants in aquatic environments. However, little is known about their effect on aquatic organisms, especially in crustaceans. In the present study, the freshwater crayfish Pacifastacus leniusculus was exposed during 21 days to environmental concentrations of sulfamethoxazole (SMX) (100 ng/L and 1 μg/L). Subsequently, the crayfish susceptibility to infection was evaluated by using White Spot Syndrome Virus (WSSV) challenge, a well-known crustacean pathogen. The median survival time of the infected crayfish exposed to 100 ng/L SMX was one day, whereas the control and the group exposed to 1 μg/L SMX survived for two and three days, respectively. In order to elucidate the effect of SMX upon the crayfish immune response, new sets of crayfish were exposed to the same SMX treatments to evaluate mRNA levels of immune-related genes which are expressed and present in hemocytes and intestine, and to perform total and differential hemocyte counts. These results show a significant down-regulation of the antimicrobial peptide (AMP) Crustin 3 in hemocytes from the 100 ng/L SMX group, as well as a significant up-regulation of the AMP Crustin 1 in intestines from the 1 μg/L SMX group. Semigranular and total hemocyte cell number were observed to be significantly lower after exposure to 100 ng/L SMX in comparison with the control group. The present study demonstrates that environmentally relevant SMX concentrations in the water at 100 ng/L led to an increased WSSV susceptibility, that may have been caused by a reduction of circulating hemocytes. Nevertheless, SMX concentrations of 1 μg/L could marginally and for a few days have an immunostimulatory effect.

Physiological and biochemical responses of Mediterranean green crab, Carcinus aestuarii, to different environmental stressors: Evaluation of hemocyte toxicity and its possible effects on immune response

Effects of natural stressors such as copper (Cu2+), temperature, hypoxia, chloroform and adrenaline on physiological and biochemical responses were investigated in the Mediterranean green crab Carcinus aestuarii from tidal shallow waters of Narta Lagoon, Albania. For this purpose, hemolymph glucose levels, total and differential hemocyte count, in normal and eye-stalked individuals, exposed to above mentioned stressors like, were assessed. In addition, lysosomal membrane stability was evaluated as biomarker of hemocyte toxicity, with possible implications on crab immune response. Hemolymph glucose levels were significantly increased in all treatment groups with 1.25-to 3.5-fold above baseline levels of 37.8 ± 2.7 mgdL−1. Response times were being manifested within 30–120 min following exposure and recovery happened within 2 h of restoration of pretreatment conditions. Total hemocyte count (THC) and differential hemocyte count (DCH) showed a significant decrease for all stressors, except for copper, were an increase of semi-granular hemocyte fraction were recorded. Meanwhile, significant reduction of neutral red retention time (NRRT), in both eyestalk-ablated and exposed animals, were recorded, indicated the loss of hemocyte lysosomal membrane integrity. The responsiveness of hemolymph blood levels to all stressors, the decrease in total hemocyte count, as well as the loss of lysosomal membrane integrity demonstrated that exposure to environmentally realistic stressors placed a heavy metabolic load on C. aestuarii, modulating their immune competence and overall physiological wellness. Overall, results suggest that monitoring cellular and biochemical parameters like hemolymph glucose titres, TCH, DHC and NRRT, may be useful and sensitive means of evaluating the crustacean's ability to cope with the wide variety of environmental stressors through modulation of the immune parameters.

Effects of DCOIT (4,5-dichloro-2-octyl-4-isothiazolin-3-one) to the haemocytes of mussels Perna perna

Bivalve molluscs rely only on an innate immune system to execute cellular and humoral processes. Haemocytes, the haemolymph circulating cells, play a major role in this type of immunity, principally regarding cellular defences. Considering that environmental pollutants can affect the immune system of invertebrates, this work evaluated the effects of the antifouling biocide 4,5-dicloro-2-n-octil-4-isotiazolin-3-ona (DCOIT) on the haemocytes of mussels Perna perna. Individuals were exposed to 0 (control), 0.1 μg L−1 and 10 μg L−1 of DCOIT for up to 96 h. The analysed parameters included: total (THC) and differential (DHC) haemocyte count, cellular viability, adhesion capacity, phagocytic activity, levels of reactive oxygen species and DNA damage. Moreover, the stress on stress (SOS) response of mussels was analysed as a general stress index. The results show that DCOIT increased the haemocyte adhesion capacity and caused a decrease in THC and in the haemocyte viability after 24 h of exposure. After 96 h of exposure, DCOIT only affected the haemocyte adhesion capacity, which was decreased by biocide exposure. Moreover, exposure to DCOIT for 96 h did not affect the capacity for air survival of mussels. These results indicate that DCOIT interferes in important parameters associated with the innate immunity of P. perna, mainly after 24 h of exposure. It is suggested that the animals were able to develop some compensatory response strategy, making them more resistant to the biocide.

DETERMINATION OF CELL TYPE AND HAEMOCYTE MORPHOMETRIC CHARACTERISTICS OF WESTERN AUSTRALIA FRESHWATER CRAYFISH (Cherax cainii) AT DIFFERENT TEMPERATURES IN VITRO

The purpose of this study was to identify and morphologically characterize the hemocyte cell types of freshwater crayfish, Cherax cainii. In addition to morphological observations using a light microscope (LM) and electron transmission microscope (TEM), a flow cytometer (FCM) is also used. Three main types of haemocyte of C. cainii were identified by LM, TEM, and FCM. Determination of haemocyte by LM based on the number, size of cytoplasmic granules and the ratio of N:C. These cells are Hyaline (HC), Small Granule (SGC), and Large Granule (LGC) cells. Three types of haemocyte were also observed by TEM based on cell and nucleus size, granule diameter, number of cytoplasmic granules per cell and N:C. Haemocyte population was successfully detected with FCM based on forward scatter (FSC) signals, versus side scattering signals/side scatter (SSC), with plot data via scatter parameter gating. Three cluster formations were observed, which were temporarily classified as SGC, LGC, and HC regions. Morphometric analysis was performed with TEM on C. cainii haemocyte to measure various cellular features. Some morphological features vary between types of haemocyte and are also affected by temperature. Total hemocyte count (THC) and differential hemocyte count (DHC) are calculated using FCM. THC increases with higher temperatures, from 1,9 x 106 /ml at 20 °C to 4.9 x 106 /ml at 30 °C. The most abundant hemocyte at all temperatures is HC, followed by SGC and LGC.

Effect of carbofuran on quantitative and qualitative alterations in haemolymph of larva of Oryctes rhinoceros L. (Coleoptera: Scarabaeidae)

Investigation to evaluate the toxicity of carbofuran pesticides on haematological parameters of third instar larvae of Oryctes rhinoceros L. Indicated alterations in total haemocyte count and differential haemocyte count for toxicity assessment. Various doses of carbofuran (0.05g, 0.010g and 0.015 g) applied on insect through oral route and its impact after 24 hours of its application revealed that various doses of carbofuran exert specific alterations in both total and differential haemocytes of insect haemolymph.

Do dietary stresses affect the immune system of Periplaneta americana (Blattaria: Blattidae)?

Stresses can be caused by multiple biotic and abiotic factors and their effects can affect both the biology and the immune system of insects. American cockroach - Periplaneta americana (Linnaeus, 1758) (Blattaria: Blattidae) -besides being an excellent model species, has great medical importance because it can act as a mechanical vector of several pathogens. This study aimed to evaluate the influence of starvation, dehydration and both stresses on weight, and total and differential haemocyte count in P. americana adults. Each specimen was isolated in glass flasks containing or not food and/or water. They were weighed periodically. Another group received water for 24 h after the end of stress period. In the immunologic bioassay, we counted their haemocytes after the final weighing. All stresses reduced the insect weight, especially when the stresses were combined. Females of the control group gained weight and males had it unaltered. Different stress conditions and time did not influence on total haemocyte count. Insects without food and water had the proportion of prohaemocytes increased and plasmatocytes decreased. This study can serve as a basis of further studies of bioecology, behaviour and the ability of resisting insecticides, besides serving as a model to studies in other insect species.

Effect of food plants on Spodoptera litura (Lepidoptera: Noctuidae) larvae immune and antioxidant properties in response to Bacillus thuringiensis infection

The larvae of Spodoptera litura (Fabricius) were reared on five host plants, Brassica oleracea, Nicotiana tabacum, Ricinus communis, Gossypium hirsutum, and Arachis hypogaea. The larvae were immunized with Bacillus thuringiensis to observe the immune response. The results of total and differential hemocyte count were increased in B. oleracea, N. tabacum, and R. communis fed S. litura larval hemolymph. Similar results were observed in the parameter of nodulation, melanization, and phenoloxidase. Total protein was higher in R. communis fed larvae. Antioxidant levels like Catalase (CAT), Superoxide dismutase (SOD), Glutathione S- transferase (GST), Peroxidase (POX), Lipid peroxidase (LPO), and Esterase (EST) was found in moreover all plant-feeding insect. High CAT activity was observed 2–6 h in R. communis, G. hirsutum, and A. hypogaea fed S. litura larval midgut and fatbody samples. Increased SOD activity in both midgut and fatbody at 2–12 h of B. oleracea, G. hirsutum, and A. hypogaea fed. GST activity was increased initially 2−6 h in G. hirsutum and A. hypogaea. Increased POX activity was observed initially in all treated groups. Highest LPO observed at 6 h in N. tabacum in both midgut and fatbody. Whereas increased EST activity was observed in N. tabacum and B. oleracea. The results of the present study shows that nature of food influence the immunity against Bt infection. This information can be very useful for incorporating biological control program for insect pest.

Effect of Manihot esculenta (Crantz) leaf extracts on antioxidant and immune system of Spodoptera litura (Lepidoptera: Noctuidae)

Abstract Nowadays, the use of chemical insecticides has led to development of resistance in insects, and causing harmful effect to non-target organisms and environment. Plant-mediated biopesticides are an alternative source for the management of insect pests. Present study focuses on lethal and sublethal effects of Manihot esculenta on immune response and antioxidant enzyme activity of Spodoptera litura larvae. The result shows that high toxicity of petroleum benzene leaf extract in S. litura larvae with LC50 of 109.89 mg/mL. FT-IR analysis of leaf extracts shows the presence of alkane, alkyl halides, misc, amines, esters and carboxylic acid. GC-MS analysis of extracts contains 10 compounds, notable among them are 9, 12-Octadecadienoic acid (10.93) and Lupeol (11.35), 4, 4, 6A, 6B, 8A, 11, 14 B-Octamethyl- (37.41). In addition, several sublethal effects of the leaf extract were also observed. There was a significant increase in the total haemocyte count and differential haemocyte count in S. litura larvae. Phenoloxidase (PO) levels, detoxification enzymes such as CytP450, GST and catalase levels were significantly increased, whereas, SOD levels were lower than the non-treated S. litura larvae. Manihot esculenta leaf extracts is an alternative to chemical insecticides for the management of S. litura larvae and may be used for the management of other agricultural insect pests.

Immune and nutritional responses of Podisus nigrispinus (Hemiptera: Pentatomidae) nymphs sprayed with azadirachtin

AbstractAzadirachtin is an efficient biopesticide, and it is considered safe for natural enemies because its mode of action is mainly by ingestion. The predator, Podisus nigrispinus Dallas, is found in various agroecosystems as a potential tool for biological control. However, there is much debate on the safety of this stinkbug if azadirachtin is used. Thus, we evaluated the cellular and humoral responses as well as biochemical parameters of the fifth‐instar nymphs of P. nigrispinus treated with azadirachtin. The insects were divided into three treatment groups and sprayed with azadirachtin (Azamax®), deltamethrin (Decis®), and water (control). After 24 h, total and differential hemocyte counts were performed. The analysis of phenoloxidase activity and quantification of nitric oxide, lipids, sugar, glycogen, and protein were performed after an interval of 24 and 72 h. Azadirachtin was found to affect the immune system of this stinkbug and decrease the number of hemocytes, especially pro‐hemocytes and oenocytoids, and it may have caused a reduction in phenoloxidase activity and nitric oxide level. In addition, the decrease in protein levels indicated the interference of azadirachtin in protein synthesis. We conclude that damage caused by the botanical insecticide has been less harmful than that caused by the synthetic insecticide, but use of azadirachtin may still interfere with biological control due to undesirable immune system damage reducing predator efficiency.

Physiological, histopathological and cellular immune effects of Pergularia tomentosa extract on Locusta migratoria nymphs

The migratory locust Locusta migratoria (Orthoptera, Acrididae) is one of the most important pests due to its extensive and severe damage to crops in large parts of Africa and Asia. Biodegradable and ecologically natural products such as botanical insecticides are emerging candidates for replacement of usually applied chemical pesticides. The crude methanolic extract of Pergularia tomentosa (PME) was investigated for their toxicity and physiological aspects on L. migratoria nymphs. Results showed that treatment of newly emerged fourth and fifth instar nymphs resulted in significant mortality and significant repellent activity with an LC50 value of 0.18 and 0.38%, respectively, after seven days of treatment. The PME toxicity was also demonstrated by histopathological changes in the alimentary canal resulting in considerable disorganization and severe damage of the caeca and proventriculus structure. The extract induced cellular immune reactions which manifested by a significant decrease in the number of the differential haemocyte counts (prohemocytes and plasmatocytes) and important cell lysis. Data of biochemical analyses showed that the PME reduced the activity of acetylcholinesterase and induced the glutathione S-transferases. The neurotoxic effect was confirmed by the histological alterations in the brain structure, particularly in the neurosecretory cells showing typical signs of cell necrosis.

Variations in hemocyte profile induced by Beauveria bassiana (Bals.) Vuill. in Periplaneta americana (L.) (Blattodea: Ectobiidae)

Hemocytes of healthy (untreated) and Beauveria bassiana (Bals.-Criv.) Vuill.-treated nymphs and adults Periplaneta americana (L.) (Blattodea: Ectobiidae) were examined with phase contrast microscopy. The total hemocyte counts (THCs) and differential hemocyte counts (DHCs) were estimated and compared between the untreated and treated nymphs and adults; significant augmentation of THC was observed in the treated 3rd, 4th, 5th instars and adults at 6 h after treatment (HAT), compared to the untreated individuals, followed by its suppression. In addition to changes in the population dynamics of prohemocytes (PRs), plasmatocytes (PLs), granulocytes (GRs) and spherulocytes (SPs), their morphology was also affected. GRs, predominant hemocytes involved in cell-mediated defense, responded to the infection differently including the formation of fine pseudopodia–like cytoplasmic extensions and, they clumped together.

Hemolymph cytology, hemocyte count, glucose, and electrolyte reference intervals in 93 Cameroon red tarantulas (Hysterocrates gigas).

Theraphosidae is a large family of tarantula spiders commonly kept as pets or display animals by zoological institutions. Interest and demand for arachnid medicine have grown over the last decade. While hemolymph analysis could be a fundamental tool for arachnid health assessment, RIs are generally lacking for arachnid species. The objectives of this study were to describe the appearance of hemocytes and establish RIs for total and differential hemocyte counts as well as hemolymph glucose and electrolyte concentrations in the Cameroon red tarantula (CRT) (Hysterocrates gigas). Ninety-three CRTs were anesthetized using isoflurane or sevoflurane, and hemolymph was collected from the heart. Hemocyte counts were performed using a hemocytometer and trypan blue stain, and differential cell counts were estimated using light microscopy and a Wright stain. Hemocytes were also characterized with transmission electron microscopy (TEM). Hemolymph glucose and electrolyte concentrations were obtained using a blood gas analyzer. The American Society for Veterinary Clinical Pathology (ASVCP) guidelines were used to calculate RIs. The most abundant hemocytes were plasmatocytes, followed by granulocytes, cyanocytes, and prohemocytes. Hemocytes were characterized by TEM except for cyanocytes. All RIs had a broad range, likely due to a combination of population heterogeneity, high biologic variability of the species, and the propensity of samples to clot. The current study is the first to establish RIs for hemolymph cytologic parameters with a sample size of over 20 individuals in any theraphosid species. RIs for CRT hemolymph biochemical analytes are also reported.

Harpalus (Pseudoophonus) rufipes as a model to study cellular and humoral immune defence strategies in coleopteran species

Carabids are of special interest as environmental quality assessment indicators of exposure to xenobiotic and for pest control. In agroecosystems, they can be exposed to a wide range of pathogens and environmental pollution exerting a stronger selection on their innate immune systems. Therefore, information on species-specific immunocompetence is necessary to complete the ecological framework of ground beetles. In this study, cellular and humoral responses were characterized in adults of Harpalus (Pseudoophonus) rufipes (De Geer, 1774) to define a baseline knowledge for future ecotoxicological studies. The circulating hemocytes were characterized by light and transmission electron microscopy and in vivo assay performed by injecting latex beads to identify phagocytizing hemocytes. Ultrastructural analyses revealed four morphologically distinct types of circulating hemocytes: prohemocytes, plasmatocytes, granular cells and oenocytoids. Differential hemocyte counts showed that plasmatocytes and granular cells were the most abundant circulating cell types and granular cells exhibited phagocytic activity following immune challenge with latex beads. Mitotic figures and non-differentiated hemocytes observed under light microscopy indicate a continuous cell turnover in the hemolymph. Melanotic nodules found 2h after the immune challenge were formed to immobilize the latex beads. Phenoloxidase (PO) assays revealed an increase of basal PO activity in hemolymph after immune system activation with lipopolysaccharide (LPS). However, the LPS-stimulated adults showed no significant variation in the lysozyme-like enzyme activity in hemolymph. Based on these results, H. rufipes displays a rapid, non-specific immune response involving cellular and humoral effectors that both sequester and clear pathogens.

Growth performance, immune response, and digestive enzyme activity in Pacific white shrimp, Penaeus vannamei Boone, 1931, fed dietary microbial lysozyme

The present study investigates the effects of the dietary microbial lysozyme (ML) as an immunostimulant, on the growth performance, some immune parameters and digestive enzyme of Pacific white shrimp, Penaeus vannamei. Six hundred shrimps were obtained and randomly allocated into four groups as follows with three replicates. The shrimps were fed diets supplemented with 0 (control), 0.5, 1, and 2 g kg−1 ML for 4 months. The results indicated that dietary supplementation of ML significantly improved final weight, weight gain, average daily weight gain rate (ADG), feed conversion rate (FCR), and feed efficiency rate (FER) compared to the control (P ˂ 0.05). However, weight gain specific growth rate (SGR) and survival rate were not significantly affected by dietary ML (P ˃ 0.05). Dietary ML had a progressive effects on some immune parameters status including total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GPX), lysozyme (LYZ), alkaline phosphatase (ALP), phenoloxidase (PO) and acid phosphatase (ACP) activity as well as differential haemocyte count (DHC) and total haemocyte count (THC), in shrimps treated with the lysozyme than untreated shrimps (P ˂ 0.05). However, feeding with ML had no significant effect on plasma malondialdehyde (MDA) level (P ˃ 0.05). Furthermore, intestinal digestive enzymes (lipase, protease, and amylase) in shrimp fed with dietary ML were significantly (P ˂ 0.05) higher than those fed with non-supplemented control basal diet. Thus, the results indicate that oral administration of ML can be recommended for shrimp feed to improve immune response as well digestive enzymes activity modulation.

Boric acid-induced immunotoxicity and genotoxicity in model insect Galleria mellonella L. (Lepidoptera: Pyralidae).

Boric acid (BA) is widely used in various industrial process and can be accessed to nontarget organisms. This study aimed to investigate the insecticidal effects of BA and its toxic activities with respect to immunologic and genotoxic effects using Galleria mellonella larvae as a model. BA concentrations (78.125-10,000 ppm) were administrated to the larvae using the feeding method. Concentration-dependent mortality was observed in all larval groups. Probit analysis revealed LC30 , LC50 , and LC70 values to be 112.4, 320.1, and 911.4 ppm, respectively. These concentrations were used in all bioassays. Drastic reductions in total hemocyte counts along with changes in differential hemocyte counts were observed following BA treatment. Cell viability assays showed dose-dependent reductions in viable cells and an increase in the necrotic and apoptotic ratios after BA treatment. However, mitotic indices of larval hemocytes did not change at all BA concentrations. The cytotoxic effect of BA led to a significant reduction in cellular immune responses such as encapsulation, melanization, and nodulation activities of treated larvae. While BA increased micronucleus ratios at the highest concentration, comet parameters indicating DNA damage increased in G. mellonella larval hemocytes at all concentrations. These report that BA suppresses the immune system of G. mellonella and also poses risks of genotoxicity at high concentrations.

The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon

Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000 pmol shrimp−1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3′,5′-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10 nmol shrimp−1. Results showed that all of the immune parameters significantly increased at 2–4 h in OA-injected shrimp except hyaline cells in 100 pmol shrimp−1-injected shrimp at 4 h, but phenoloxidase activity per granulocyte significantly decreased at 2–4 h. However, these had returned to saline control levels after receiving OA for 8 h except differential haemocyte count and phenoloxidase activity per granulocyte for 16 h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10 nmol shrimp−1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30–60 min and 30 min, and cAMP concentration [cAMP]i) at 5–15 min and 15 min, respectively. However, [Ca2+]i at 50–60 min, and [cAMP]i at 30–60 min returned to saline control when the shrimp received OA at 10 nmol shrimp−1, and at 1 and 10 nmol shrimp−1, respectively. These results suggest that OA administration by injection at ≤1000 pmol shrimp−1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.