Partial inhibition of hemocyte agglutination by Lathyrus odoratus lectin in Crassotrea virginica infected with Perkinsus marinus

Quantitative determinations of agglutination of hemocytes from oysters, Crassostrea virginica, by the Lathyrus odoratus lectin at five concentrations revealed that clumping of hemocytes from oysters infected with Perkinsus marinus is partially inhibited. Although the nature of the hemocyte surface saccharide, which is not D(+)-glucose, D(+)mannose, or alpha-methyl-D-mannoside, remains to be determined, it may be concluded that this molecule also occurs on the surface of P. marinus. It has been demonstrated that the panning technique (Ford et al. 1990) is qualitatively as effective for determining the presence of P. marinus in C. virginica as the hemolymph assay method (Gauthier & Fisher 1990).

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals

Hemocyte types and total and differential counts in unparasitized and parasitized Anastrepha obliqua (Diptera, Tephritidae) larvae

Os tipos de hemócitos e as contagens total e diferencial foram estudados em larvas parasitadas e não parasitadas de Anastrepha obliqua pertencentes ao início e ao final da terceira fase. em ambas as fases do desenvolvimento, tanto em larvas parasitadas quanto nas não parasitadas, foram observados pró-hemócitos, plasmatócitos, granulócitos, adipo-hemócitos, esferulócitos e oenocitóides. A presença de divisões mitóticas indica os pró-hemócitos como células-tronco. Pró-hemócitos, plasmatócitos e granulócitos são as células mais numerosas na hemolinfa de A. obliqua. Foi observada diferença no número total de hemócitos entre larvas parasitadas e não parasitadas apenas no final da terceira fase.

Insect/Bt-cotton interactions: Are immunological variables and hemocyte ultrastructure in Podisus nigrispinus (Hemiptera: Pentatomidae) affected?

Recent studies have shown that ingestion by the army worm Spodoptera frugiperda of Cry1Ac toxin from Bt cotton promotes histochemical and ultrastructural changes in the digestive cells of the predatory pentatomid bug Podisus nigrispinus. Therefore, mindful of the changes in the midgut of the predator, which represents the first line of defence in this insect, our aim was to test the hypothesis that the consumption of Bt cotton-fed S. frugiperda by P. nigrispinus might lead to alterations in components of the immune system of P. nigrispinus. The Cry1Ac toxin level in the leaves of Bt cotton, nitric oxide, phenoloxidase activity, and total proteins were quantified by ELISA. Total and differential hemocyte counts were evaluated, and hemocyte ultrastructure analysis was undertaken. We found that ingestion of the prey fed daily with approximately 23 ± 0.70 ng g-1 Cry1Ac by wet weight of leaves, and expressed by the Bt cotton, induces small ultrastructural changes in the predator's granulocytes and plasmatocytes. However, these changes did not affect the total number and differential and humoral variables analyzed for the bug's hemocytes. © 2013 Copyright Taylor and Francis Group, LLC.

A calreticulin-like protein from endoparasitoid venom fluid is involved in host hemocyte inactivation

During oviposition, most endoparasitoid wasps inject maternal factors into their hosts to interfere with host immune reactions and ensure successful development of their progeny. Since encapsulation is a major cellular defensive response of insects against intruding parasites, parasitoids have developed numerous mechanisms to suppress the host encapsulation capability by interfering with every step in the process, including recognition, adherence and spreading. In previous studies, components of Cotesia rubecula venom were shown to inhibit melanization of host hemolymph by interfering with the prophenoloxidase activation cascade and facilitate expression of polydnavirus genes. Here we report the isolation and characterization of another venom protein with similarity to calreticulin. Results indicate that C rubecula calreticulin (CrCRT) inhibits hemocyte spreading behavior, thus preventing encapsulation of the developing parasitoid. It is possible that the protein might function as an antagonist competing for binding sites with the host hemocyte calreticulin, which mediates early-encapsulation reactions. (c) 2005 Elsevier Ltd. All rights reserved.

Persistence and expression of Cotesia congregata polydnavirus in host larvae of the tobacco hornworm, Manduca sexta

The gregarious braconid wasp Cotesia congregata parasitizes host larvae of Manduca sexta, and several other sphingid species. Parasitism induces host immunosuppression due to the disruptive action of the wasp's polydnavirus (PDV) on host blood cells. During the initial stages of parasitism, these cells undergo apoptosis followed by cell clumping, which clears the hemolymph of a large number of cells. In this study, the persistence and expression of Cotesia congregata PDV (CcPDV) were examined using Southern and Nor-them blots, respectively. Digoxygenin-labelled total polydnaviral DNA was used to probe genomic DNA isolated from fat body and brains of hosts with emerged wasps taken 6 days following egress of the parasitoids, and significant cross-hybridization between the host fat body genomic DNA with viral DNA was seen. Thus, the virus persists in the host for the duration of parasitism. even during the post-emergence period, and may even be integrated in the host caterpillar DNA. Viral gene expression was examined using Northern blots and probes to the Cotesia rubecula CrV1 homolog, and the CrV1-like mRNAs were expressed as early as 4 h post-parasitization for at least 72 h and faint hybrization is even seen at the time the wasps eclose. In contrast, in Pieris rapae larvae the CrV1 transcript is expressed only for a brief time, during which time hemocyte function is disrupted. The effect is transitory, and hemocytes regain their normal functions after the parasites emerge as first instars. The genome of CcPDV contains one copy of the CrV1-like homolog as shown on Southern blots of viral genomic DNA. In conjunction with our earlier studies of the PDV-encoded early protein 1, the current work suggests multiple viral transcripts are produced following parasitization of the host. and likely target host hemocytes to induce their apoptosis, thereby preventing encapsulation of the parasitoid's eggs. Whether viral DNAs are integrated in the host's genomic DNA remains to be proven, but our results provide preliminary evidence that viral DNAs are detected in the host's fat body cells examined at the time of wasp ernergence and several days later. (C) 2003 Elsevier Science Ltd. All rights reserved.

The hemocytes of Panstrogyllus Megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduvidae)

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrogylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, espcieally rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER).They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrondensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrondense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasme is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/cubic millimeter of hemolymph in the 4th intar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.

Aspects of classification of Hemiptera hemocytes from six triatomine species

The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types wereidentified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cytocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstrongylus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work wicah are discussed in detail.

The effects of feeding Karlodinium veneficum (PLY # 103; Gymnodinium veneficum Ballantine) to the blue mussel Mytilus edulis

The effects of exposure to the type species for Karlodinium veneficum (PLY # 103) on immune function and histopathology in the blue mussel Mytilus edulis were investigated. Mussels from Whitsand Bay, Cornwall (UK) were exposed to K. veneficum (PLY # 103) for 3 and 6 days. Assays for immune function included total and differential cells counts, phagocytosis and release of extra cellular reactive oxygen species. Histology was carried out on digestive gland and mantle tissues. The toxin cell quota for K. veneficum (PLY #103) was measured by liquid chromatography-mass spectrometry detecting two separable toxins KvTx1 (11.6 ± 5.4 ng/ml) and KvTx2 (47.7 ± 4.2 ng/ml). There were significant effects of K. veneficum exposure with increasing phagocytosis and release of reactive oxygen species following 6 days exposure. There were no significant effects on total cell counts. However, differential cell counts did show significant effects after 3 days exposure to the toxic alga. All mussels produced faeces but not pseudofaeces indicating that algae were not rejected prior to ingestion. Digestive glands showed ingestion of the algae and hemocyte infiltration after 3 days of exposure, whereas mantle tissue did not show differences between treatments. As the effects of K. veneficum were not observed in the mantle tissue it can be hypothesized that the algal concentration was not high enough, or exposure long enough, to affect all the tissues. Despite being in culture for more than 50 years the original K. veneficum isolate obtained by Mary Parke still showed toxic effects on mussels.

Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.

Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: Histopathology, immune responses, and recovery

Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods. Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense. No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.

Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila

The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmiter of the trematode Schistosoma mansoni in Brazil, were examined by ligth and transmission electron microscopy (TEM). Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature), poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite.

Sequential histological changes in Biomphalaria glabrata during the course of Schistosoma mansoni infection

Biomphalaria glabrata, highly susceptible to Schistosoma mansoni, were seen to shed less and less cercariae along the time of infection. Histological examination kept a close correlation with this changing pattern of cercarial shedding, turning an initial picture of no-reaction (tolerance) gradually into one of hemocyte proliferation with formation of focal encapsulating lesions around disintegrating sporocysts and cercariae, a change that became disseminated toward the 142nd day post miracidial exposure. Findings were suggestive of a gradual installation of acquired immunity in snails infected with S. mansoni.