Granular Hemocytes Research Articles

Molecular cloning, characterization and expression analysis of a C-type lectin (AJCTL) from the sea cucumber Apostichopus japonicus

For the sea cucumber Apostichopus japonicus, a C-type lectin (AJCTL) was identified using rapid amplification of cDNA ends (RACE) PCR techniques. The full-length cDNA of AJCTL is composed of 710bp with a 618bp open reading frame (ORF) that encodes a polypeptide of 205 amino acids with a N-terminal signal peptide and a C-terminal C-type lectin domain (CTLD). The calculated molecular mass of the whole protein is 22.5kDa and its predicted isoelectric point is 5.59. AJCTL belongs to the group VII of regulatory proteins and it might function as a Ca2+-dependent monosaccharide binding lectin specifically and recognizing mannose-type ligands. In situ hybridization demonstrated that the expression of AJCTL was located in the body wall, longitudinal muscles, intestinum and respiratory tree. This became apparent especially in the cytoplasm of epidermal cells and granular haemocytes. Real-time PCR data suggested that AJCTL was mostly synthesized in the longitudinal muscles and intestinum and less pronounced in the respiratory tree and body wall of adults. After 12h stimulation by Vibrio harveyi, at increasing bacterial concentration gradient, the expression of AJCTL in sea cucumber increased as well. This indicated that CTL is related to an innate immune response.

A Kazal type serine proteinase SPI Pm2 from the black tiger shrimp Penaeus monodon is capable of neutralization and protection of hemocytes from the white spot syndrome virus

A Kazal type serine proteinase SPI Pm2 is abundantly expressed in the hemocytes and shown to be involved in innate immune response against white spot syndrome virus (WSSV) in Penaeus monodon. The SPI Pm2 is expressed and stored in the granules in the cytoplasm of semigranular and granular but not the hyaline hemocytes. Upon WSSV challenge and progression of infection, the SPI Pm2 was secreted readily from the semigranular and granular hemocytes. The more they secreted the SPI Pm2, the less they were distinguishable from the hyaline cells. The WSSV-infected cells were either semigranular or granular hemocytes or both and depleted of SPI Pm2. The rSPI Pm2 was able to temporarily and dose-dependently neutralize the WSSV and protect the hemocytes from viral infection judging from the substantially less expression of WSSV late gene VP28. The antiviral activity was very likely due to the binding of SPI Pm2 to the components of viral particle and hemocyte cell membrane.

The use of probiotics during the nursery rearing of the pink shrimpFarfantepenaeus brasiliensis(Latreille, 1817) in a zero exchange system

The present work evaluated the use of probiotics during the nursery rearing of the pink shrimp Farfantepenaeus brasiliensis, in a zero exchange aerobic heterotrophic culture system during 30 days. Three replicate tanks were randomly assigned to the following treatments: (1) Bacillus spp. mixture (Sanolife Pro--W®), (2) Bacillus sp., Enterococcus sp., Lactobacillus spp. mixture (Biomin Start-grow®), (3) Bacillus cereus var. toyoi and (4) control treatment (without probiotic addition). Bacteriological analysis monitored the abundance of presumptive Vibrio spp. in the water of experimental tanks. For the immunological analysis, shrimp haemolymph was collected to determine the granular haemocyte count and total protein concentration. Results showed that mean final weight and specific growth rate of shrimp in the probiotic treatments were significantly higher. Furthermore, shrimp reared in the probiotic treatments showed higher levels of total protein and granular haemocyte. The bacteriological analysis showed that the concentration of Vibrio spp. measured in probiotic treatment tanks was lower than that recorded in the control tanks.

Alpha2-macroglobulin from an Atlantic shrimp: Biochemical characterization, sub-cellular localization and gene expression upon fungal challenge

In this study, we report on the isolation and characterization of an alpha2-macroglobulin (α2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the α2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that α2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the α2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70–90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus α2M made it possible to demonstrate that α2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay). Quantitative real-time PCR (qPCR) analysis showed that α2M mRNA transcripts significantly increased 24 h after an experimental infection with the shrimp pathogen Fusarium solani and it returned to the basal levels at 48 h post-injection. This is the first report on a α2M characterization in an Atlantic penaeid species and its expression profile upon a fungal infection.

Losing the battle against fungal infection: Suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2×103, 2×106 or 2×108 conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia.

BackgroundMost of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.Methodology/Principal FindingsIn A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×103 Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p<0.001).Conclusions/SignificanceThe characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.

Organic selenium supplementation promotes shrimp growth and disease resistance to Taura syndrome virus

To examine the effect of selenium (Se) supplementation on shrimp growth and disease resistance after Taura Syndrome virus (TSV) challenge, groups of Pacific white shrimp (Penaeus vannamei) from a strain known to be susceptible to TSV were fed 3 diets: (1) standard commercial shrimp feed (0.58 ppm Se content) without Se supplementation (standard diet group); (2) standard commercial feed plus 0.3 ppm inorganic Se (Inorg Se-group); and (3) standard commercial feed plus 0.3 ppm organic Se (Org Se-group) over a 5-week period. Samples (3 shrimp) were collected from all replicates before feeding (pre-feeding or week 0) and at the 1st, 2nd, and 3rd weeks post feeding to determine weight, length, and number of granular haemocytes. After the feeding trial, the remaining shrimp were used for a final TSV challenge test. Significant differences in weight between groups (P < 0.05) were found on the fifth week post feeding in which the average weight of shrimp fed the standard feed was 7.2 1.2 g compared to 6.1 1.2 g in the inorg Se-group and 9.0 1.1 g in the org Se-group. Significant increase in the number of total haemocytes and granular haemocytes (GHs) were found only in the org Se-group on the third week post feeding. On day 6 post TSV challenge, the number of survivors in the org Se-group (66.7%) was also significantly higher than those of the inorg Se- (35.5%) and standard diet- (13.3%) group. Nested RT-PCR analysis showed that the number of shrimp with severe infection in the org Se-group (2/5) was less than the inorg Se- (4/5) and standard diet-group (4/4). The RT-PCR results were similar to those observed by histological examination. The number of total haemocytes and GHs isolated from survivors of the standard diet-group were significantly lower than those isolated from survivors of the inorg Se- or org Se-group. The present results indicated that organic Se supplementation in shrimp feed could improve shrimp growth and survival after an experimental challenge with TSV.

Sadaaki Iwanaga: discovery of the lipopolysaccharide- and -1,3-D-glucan-mediated proteolytic cascade and unique proteins in invertebrate immunity

Horseshoe crab haemolymph contains a single type of cells, granular haemocytes, which are extremely sensitive to bacterial lipopolysaccharides (LPS) and lead to haemolymph coagulation. Sadaaki Iwanaga isolated protease zymogens from the haemocytes and reconstituted LPS and beta-1,3-d-glucans-mediated haemolymph coagulation. This led to the first discovery of a proteolytic cascade triggered by pathogen-associated molecular patterns, an important milestone for studies on invertebrate innate immunity. Moreover, he separated components derived from haemocyte granules and haemolymph plasma, and consequently identified unique defense molecules, such as lectins, serpins, cystatins, antimicrobial substances and substrates for transglutaminase. Through steady and persistent studies on the horseshoe crab host defense system, he made great progress in the field. Now we know that LPS-induced haemocyte exocytosis leads not only to coagulation but also activates a sophisticated immune response network that coordinately induces pathogen recognition, elimination and wound healing.

The mast cell: an evolutionary perspective

This review article is an attempt to trace the evolution of mast cells (MCs). These immune cells have been identified in all vertebrate classes as single-lobed cells containing variable amounts of membrane-bound secretory granules which store a large series of mediators, namely histamine, proteases, cytokines and growth factors. Other MC features, at least in mammals, are the c-kit receptor for the stem cell factor and the high-affinity receptor, FcepsilonRI, for immunoglobulin E (IgE). The c-kit receptor also has been identified in fish MCs. The FcepsilonRI receptor seems to be a more recent acquisition in MC phylogenesis given that IgE originated in mammalian species. Tryptase and histamine have also been recognized in MCs of teleost fish. Thus, a cell population with the overall characteristics of higher vertebrate MCs is identifiable in the most evolutionarily advanced fish species. Two potential MC progenitors have been identified in ascidians (urochordates which appeared approximately 500 million years ago): the basophil/MC-like granular haemocyte and the test cell. Both contain histamine and heparin, and provide defensive functions. Some granular haemocytes in Arthropoda also closely approximate the ultrastructure of modern MCs. The origin of MCs is probably to be found in a leukocyte ancestor operating in the context of a primitive local innate immunity and involved in phagocytic and killing activity against pathogens. From this type of defensive cell, the MC phylogenetic progenitor evolved into a tissue regulatory and remodelling cell, which was incorporated into the networks of recombinase activating genes (RAG)-mediated adaptive immunity in the Cambrian era, some 550 million years ago. Early MCs probably appeared in the last common ancestor we shared with hagfish, lamprey and sharks about 450-500 million years ago.

Flow Cytometric Analysis of Defense Functions of Hemocytes from the Penaeid Shrimp,Penaeus vannamei

Morphology and functions of hemocytes from Penaeus vannamei were analyzed by flow cytometry (FCM) on the basis of cellular structures and incorporation of fluorescent markers. In shrimps there were three types of circulating hemocytes (hyalinocytes, semi-granulocytes, and granulocytes) identified by FCM and light microscopy. Analysis of hemocytes after propidium iodide staining indicated that the majority of hemocytes were alive after been collected in the physiological anticoagulant. The phagocytic activity level of hemocytes was analyzed using fluorescent beads and the shrimp hemocytes showed different phagocytic activity that could be inhibited by sodium azide. Respiratory burst activity, which was measured using 2′,7′-dichlorofluorescein diacetate, can be stimulated by phorbol-12-myristate-13-acetate. Mitochondrial membrane potential was probed according to the accumulation of 3′,3′-dihexyloxacarbocyanine iodide in the mitochondrial matrix. The data showed more granular cells displayed higher fluorescence intensity than less granular hemocytes. FCM can rapidly, accurately, and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the shrimp immune system. In addition, such parameters can be used in later experiments to measure defense mechanisms and the modulation of shrimp response to pathogenic agents and environmental stress.

Flow Cytometric Characterization of Freshwater Crayfish Hemocytes for the Examination of Physiological Status in Wild and Captive Animals

Enumeration of invertebrate hemocytes is a potentially powerful tool for the determination of physiological effects of extrinsic stressors, such as hypoxia, disease, and toxicant exposure. A detailed flow cytometric method of broad application was developed for the objective characterization and enumeration of the hemocytes of New Zealand freshwater crayfish Paranephrops planifrons for the purpose of physiological health assessment. Hemocyte populations were isolated by flow cytometric sorting based on differential light scatter properties followed by morphological characterization via light microscopy and software image analysis. Cells were identified as hyaline, semigranular, and granular hemocytes based on established invertebrate hemocyte classification. A characteristic decrease in nuclear size, an increase in granularity between the hyaline and granular cells, and the eccentric location of nuclei in granular cells were also observed. The granulocyte subpopulations were observed to possess varying degrees of granularity. The developed methodology was used to perform total and differential hemocyte counts from three lake populations and between wild and captive crayfish specimens. Differences in total and differential hemocyte counts were not observed among the wild populations. However, specimens held in captivity for 14 d exhibited a significant 63% reduction in total hemocyte count, whereas the relative hemocyte proportions remained the same. These results demonstrate the utility of this method for the investigation of subacute stressor effects in selected decapod crustaceans.

Structural alterations in the male reproductive system of the freshwater crayfish, Cherax quadricarinatus (Decapoda, Parastacidae)

No diseases affecting reproductive performance have been previously reported in freshwater crayfishes. This study aims to characterise one reproductive system abnormality found in males of Cherax quadricarinatus reared in captivity. Fifteen adult males of C. quadricarinatus (70–110 g) were purchased from San Mateo S.A. farm (Entre Ríos, Argentina) each season during 2007. Macroscopic analysis showed that 26.6% of the animals sacrificed in winter presented brownish distal vasa deferentia. Histological analysis showed different levels of structural abnormality in the epithelium of the vasa deferentia and spermatophore. Granular and hyaline haemocytes were identified within the vasa deferentia but no significant differences were found in the sperm count between normal and brownish vas deferens. Histological analysis of the crayfishes sacrificed in autumn also showed these modifications in 22% of the animals, however, they did not show the brownish colour under macroscopic analysis. The similarities between the male reproductive system syndrome in shrimps and the abnormalities found in C. quadricarinatus are notable. An unspecific response to thermic stress is a possible explanation of these structural alterations.

Time-course analysis of peroxinectin mRNA in the shrimp Litopenaeus vannamei after challenge with Vibrio campbellii

Peroxinectin (Pox), which promotes cell adhesion and encapsulation of bacteria in crustaceans, is synthesized in granular and semigranular hemocytes. In this study, real-time PCR was used to quantify Pox transcripts in individual tissues of the Pacific white shrimp, Litopenaeus vannamei, over 48 h following injection of a sublethal dose of the shrimp pathogen Vibrio campbellii. The resulting data were used to infer the movements of hemocytes among the tissues in response to bacterial challenge. Over all times and treatments, Pox transcripts (ng total RNA) −1 varied by orders of magnitude among individual tissues, such that circulating hemocytes » gills » heart › lymphoid organ › hepatopancreas ≈ muscle. Relatively low constitutive expression of Pox in the lymphoid organ compared to circulating hemocytes, gills, and heart supports a primary role for this organ in bacteriostasis and degradation, rather than encapsulation of invasive bacteria. Numbers of Pox transcripts increased significantly at the injection site within 4 h and remained significantly elevated for 48 h, consistent with a rapid and sustained recruitment of hemocytes to the site of injection. Transcripts increased significantly in the gill but not in other tissues over the time-course of this experiment. These expression data reinforce the role of the gill in trapping and encapsulating invasive bacteria as a primary strategic focus during the early phase of the crustacean immune response and, by comparison with earlier studies of lysozyme expression in the same tissues, suggest differential roles for various tissues in a successful immune response.

Activation of immunological responses in Litopenaeus setiferus hemocytes by a hemocyanin like-lectin

In order to identify the specific role of humoral lectins in crustaceans' immune response, we evaluated the effect of the 291 kDa hemocyanin-like lectin from Litopenaeus setiferus (LsL) on its own hemocytes. LsL binds 5.6% of circulating non-activated hemocytes, but is able to recognize 85% of total adhered (activated) hemocytes, showing filipodia and pseudopodia, inducing the production of reactive oxygen intermediates (ROIs) in a time- and dose-dependent manner (100 ng/10 5 cells/20 min). This effect was inhibited specifically by N-acetylated sugars (NeuAc, GalNAc or GlcNAc), as well as by superoxide dismutase (SOD), acetylsalicylic, and mefenamic acid. In hemocytes, LsL also induced higher ROIs production than Zymozan ( p > 0.05). The receptor for LsL in the hemocytes membrane is a glycoprotein of 61 and 52 kDa per subunit, as determined by western blot. Our results suggest that LsL participates in the activation of oxidative immune responses of granular hemocytes induced by NADPH and other oxidative pathways through a specific membrane receptor.

Purification and characterization of antibacterial proteins from granular hemocytes of Indian mud crab, Scylla serrata.

Marine invertebrates depend upon antimicrobial peptides (AMPs) as a major component of innate immunity, as they are rapidly synthesized and diffuse upon pathogen invasion. In this study, we report the identification and characterization of a 11 kDa antimicrobial protein, which we name SSAP (for Scylla serrata antimicrobial protein), from granular hemocytes of the mangrove crab S. serrata. The protein is highly similar to scygonadin, a male-specific AMP isolated from the ejaculatory duct of S. serrata. SSAP was isolated using various chromatographic techniques, viz. ion-exchange, ultra filtration and RP-HPLC, and demonstrated antibacterial activity against Gram positive and Gram negative bacteria. Full length mRNA encoding SSAP was amplified using a combination of RT-PCR and RACE. The nucleotide sequence revealed a full-length ORF of 381 bp coding for a preprotein of 126 amino acids comprising a signal peptide of 24 amino acids and a mature protein of 102 amino acids with a predicted mass of 11435 Da and pI of 5.70. Unlike scygonadin, SSAP is expressed in several tissues of both male and female crabs, as evidenced by RT-PCR, Northern and Western blot analyses. The study suggests that SSAP might be an isoform or a variant of scygonadin and might play an important role in regulating the immunity of the crab upon microbial infection.

Cellular localization of lead using an antibody‐based detection system and enzyme activity changes in the gills and digestive gland of the blue mussel Mytilus edulis

Marine organisms are continuously exposed to heavy metals in their environment. Bivalve mollusks such as the blue mussel Mytilus edulis accumulate high levels of heavy metals effecting cellular homeostasis and functions. Lead (Pb) exposure (2.5 mg/L of lead (II) nitrate for 10 d) and depuration (10 d in clean seawater) experiments were conducted to study its intracellular fate in the gills and digestive gland of M. edulis. For this purpose, an antibody-based detection method for ultrastructural localization and a subcellular fractionation approach for chemical analysis of Pb were used. In addition, effects of Pb on enzyme activities involved in oxyradical scavenging, such as the conjugative enzyme glutathione-S-transferase and the antioxidative enzyme catalase, were determined. The ultrastructural studies showed that Pb was mainly detected in lysosomes of gill epithelial cells and digestive cells. Lead was also detected in cell nuclei and granular hemocytes. Higher metal concentrations were measured by chemical analysis in subcellular fractions of the gills compared to those of the digestive gland. Increased activities of glutathione-S-transferase were found in gills after exposure and remained elevated during the depuration period, whereas glutathione-S-transferase activity remained unaffected in the digestive gland. Catalase activities showed no changes after Pb exposure, neither in the gills nor in the digestive gland. We conclude that gill cells are major sites of uptake and accumulation for dissolved Pb and are involved in sequestration and detoxification of this metal in M. edulis.

Hematological changes in white spot syndrome virus-infected shrimp, Fenneropenaeus chinensis (Osbeck)

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifestations of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and disintegrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.

Evaluation of hemato-immunological parameters during the reproductive cycle of the scallop Nodipecten nodosus in association with a carotenoid-enriched diet

The cultivation of scallops Nodipecten nodosus is a promising activity emerging in Brazil. The purpose of this study was to characterize the immune system of N. nodosus and evaluate the modulation of some hemato-immunological parameters during the reproductive cycle, in association with an astaxanthin-enriched diet. It was hypothesized that a supplementation on astaxanthin could enhance scallop immune system and minimize stress of reproduction. Scallops were separated in different groups: juveniles (J), adults (A), sexually mature (M), and recently spawned (S) animals. The last two groups were fed standard (M and S) or astaxanthin-enriched (Ma and Sa) diets. Scallop hemolymph contained two hemocyte populations: hyaline (HH) and granular hemocytes (GH). Antimicrobial peptides, similar to mussel defensins and mytilins, were found by immunodetection within the GH granules, even though the scallop hemolymph did not exhibit significant antimicrobial activity against different bacteria, including marine vibrios. Scallop hemocytes were able to phagocytose zymosan and produce reactive oxygen intermediates (ROI–NBT reduction). The number of circulating hemocytes (Neubauer chamber) varied from 12 to 26.106 cells mL−1, and the GH was always the predominant cell type (67–99%). The plasma of N. nodosus contained lectins, specific to galactose and sialoconjugates, and their agglutinating activity was partially calcium-dependent. A phenoloxidase (PO) activity (146–446 U min−1 mg−1) was observed in the scallop hemolymph. However, this activity was not induced by trypsin or components of microorganism surface, and was strongly enhanced by alkaline pH (≥8.5). The total protein concentration of the plasma varied from 240 to 660 μg mL−1. In general terms, all examined hemato-immunological parameters (hemograms, superoxide anion production, PO activity, lectin titers and total protein concentration) had a similar profile during all the scallop reproductive cycle. Their levels increased significantly from juveniles to adults (except PO activity), and declined markedly (immune depletion) in the sexually mature scallops. After spawning, the animals had a tendency to recover the standard levels of their immune parameters. Apparently, the astaxanthin-enriched diet had no effect on the tested immune parameters except for a slight influence on the scallop immune-oxidative reactions (ROI production). The results obtained in this study suggested the occurrence of a general immune depletion in the sexually mature scallops, confirming that the reproductive stage is a critical period in scallop life.

Effect of sweet flag rhizome oil ( Acorus calamus) on hemogram and ultrastructure of hemocytes of the tobacco armyworm, Spodoptera litura (Lepidoptera: Noctuidae)

Effect of the essential oil of Acorus calamus L. rhizomes, was studied on hemocytes of the tobacco armyworm, Spodoptera litura. The oil was administered in oral application at concentration of 500 and 1000 ppm to last instar larvae of S. litura and its effect on ultrastructure of hemocytes and hemogram was evaluated. The oil was administered in topical application at 250 μg dose to pupae to ascertain its effect on total and differential hemocyte counts. At both scanning (SEM) and transmission electron microscopic (TEM) levels, the major effect of oil treatment was observed on plasmatocytes (PLs) and granular hemocytes (GRs). SEM study revealed that the cytoplasmic projections of granular hemocytes were reduced, while the filopods of plasmatocytes remained unaffected. The vacuolization in the cytoplasm and degeneration of the organelles in both plasmatocytes and granular hemocytes was observed by TEM. However, no such deformities were observed in prohemocytes (PRs), spherulocytes (SPs), and oenocytoids (OEs). A concentration-dependent decrease has been observed in the larval body weight and hemolymph volume (HV), 24–72 h after treatment. In comparison to the controls, the maximum percentage growth inhibition (GI) was recorded to be 58.28 and 66.48, respectively at 500 and 1000 ppm after 72 h treatment. Similarly after 72 h treatment, the percentage reduction in hemolymph volume was 61.38 and 69.05, respectively at 500 and 1000 ppm. Total hemocyte count (THC), in larvae computed from five recorded hemocyte types viz. PRs, PLs, GRs, SPs and OEs, decreased only after 48–72 h of treatment. The maximum decrease in THC was recorded to be 29.15 and 49.05% at 500 and 1000 ppm, respectively, after 72 h of treatment. There was continuous decline in THC in pupae after 24–72 h treatment. DHC study revealed that both the concentrations of oil in 6th instar larvae of S. litura caused a decrease in PRs, PLs and SPs and increase in GRs and OEs after 24–72 h of treatment. Since A. calamus oil treatment causes the injury to both PLs and GRs and also affects the hemogram, it can be inferred that cellular defence reactions of S. litura are impaired.

Phenoloxidase-associated cellular defence in the Sydney rock oyster, Saccostrea glomerata, provides resistance against QX disease infections

The enzyme phenoloxidase is a critical component of the immunological defence of invertebrates. Previously, we have shown that the activity of phenoloxidase in Sydney rock oysters ( Saccostrea glomerata) correlates with the severity of QX disease outbreaks. The aetiological agent of QX disease is the opportunistic protozoan parasite, Marteilia sydneyi. In this study, we examined the response of oyster haemocytes to challenge with M. sydneyi. Granular haemocytes were able to rapidly phagocytose parasite sporonts. Phagocytosis stimulated intracellular associated phenoloxidase activity that led to the complete melanisation of phagosomes. Significant differences in phagocytic indexes and phenoloxidase activities were observed between oysters selected for resistance to QX disease (QXR) and non-selected wild-type oysters. The data suggest that phagocytosis and cellular melanisation are critical defensive responses of Sydney rock oysters infected with M. sydneyi.