Identification of reference markers for characterizing honey bee (Apis mellifera) hemocyte classes.

Cell mediated immunity of the honey bee (Apis mellifera) involves the activity of several hemocyte populations, currently defined by morphological features and lectin binding characteristics. The objective of the present study was to identify molecular markers capable of characterizing subsets of honey bee hemocytes. We developed and employed monoclonal antibodies with restricted reactions to functionally distinct hemocyte subpopulations. Melanizing cells, known as oenocytoids, were defined by an antibody to prophenoloxidase, aggregating cells were identified by the expression of Hemolectin, and phagocytic cells were identified by a marker expressed on granulocytes. We anticipate that this combination of antibodies not only allows for the detection of functionally distinct hemocyte subtypes, but will help to further the exploration of hematopoietic compartments, as well as reveal details of the honey bee cellular immune defense against parasites and microbes.

Implementation of various approaches to study the prevalence, incidence and progression of disseminated neoplasia in mussel stocks.

Marine mussel production is of substantial economic interest in numerous coastal areas worldwide, making crucial the study of pathologies that affect them. Disseminated neoplasia (DN) has recently been suggested to be linked to blue mussel, Mytilus edulis, mortality outbreaks observed in France since 2014, although the evidence remains indirect. In order to improve DN detection and monitoring, we compared the sensitivity of four diagnostic tools, namely haemocytology, histology, flow cytometry, and genetics. Haemocytological examination gave the best results in sensitivity and had the advantage of being non-invasive, allowing disease progression to be followed in affected mussels. Using this approach, we showed that DN progression is usually slow, and we provide evidence of remission events. We observed a high diversity of forms and mitotic features of neoplastic cells located in the vesicular connective tissue but rarely in the haemolymph. Circulating cells occur as four main types but are homogenous in morphology and DNA content within a single individual. Polyploidy proved very high, from 8 N to 18 N. Genetic analysis of haemolymph DNA showed that a Mytilus trossulus genetic signal was associated with almost all the DN cases here diagnosed by haemocytological examination, regardless of the DN type. This result corroborates DN is a transmissible cancer that first originated in a M. trossulus host and subsequently crossed into M. edulis. No pre-neoplastic conditions were detectable. The prevalence of the disease was quite low, which, together with the low morbidity observed in the lab, suggest DN is unlikely to be the direct cause of mortality outbreaks in France.

Headcase is a Repressor of Lamellocyte Fate in Drosophila melanogaster.

Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.

Dietary Protein and Carbohydrates Affect Immune Function and Performance in a Specialist Herbivore Insect (Manduca sexta).

Nutrition structures ecology and evolution across all scales of biological organization. It is well known that nutrition can have direct effects on performance and fitness, but indirect effects on physiological systems that mediate biotic interactions have been studied less frequently. Here, we focus on the interaction between nutrition, performance, and the immune system in a specialist herbivorous insect, Manduca sexta. We used a conceptual framework in nutritional ecology (the geometric framework) to examine how changes in diet quality affect aspects of the immune system used for defense against parasitoids. We raised caterpillars throughout their entire larval development on five different experimental diets that varied in protein and carbohydrate content and measured five aspects of the immune system: encapsulation, phenoloxidase activity, prophenoloxidase activity, total hemolymph protein, and hemocyte density. Overall, different parts of the immune function varied in response to interactions between carbohydrates, protein, and intake, but protein reductions had the largest impacts-mostly detrimental. In addition, our data suggest that diet quality mediates the relationship between performance (growth and survival) and immune function, as well as trade-offs among different components of immune function. Our work is the first to examine the interplay between nutrition, performance, and immune function with the geometric framework in a specialist insect herbivore.

Development of a primary culture system for haematopoietic tissue cells from Cherax quadricarinatus and an exploration of transfection methods.

Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2'-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells.

Clusterin protects neurons against intracellular proteotoxicity.

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.

The TEAD family transcription factor Scalloped regulates blood progenitor maintenance and proliferation in Drosophila through PDGF/VEGFR receptor (Pvr) signaling.

The Drosophila lymph gland is a well-characterized hematopoietic organ in which a population of multipotent stem-like progenitors is maintained by a combination of signals from different cellular populations within the organ. The lymph gland serves as an ideal model both for the interrogation of signaling mechanisms involved in progenitor maintenance as well as a tool for the identification of novel regulatory mechanisms in the highly conserved process of hematopoiesis. Here, we demonstrate a requirement for the TEAD transcription factor Scalloped in the maintenance and proliferation of hematopoietic progenitors. We have characterized a novel population of hemocytes in the early lymph gland identified by the expression of Hand, Scalloped, and the PVR ligand PVF2. In this unique population, we show that Scalloped maintains PVF2 expression, which is required for hemocyte proliferation and achievement of normal lymph gland size. We further demonstrate that STAT signaling marks actively proliferating hemocytes in the early lymph gland, and inhibition of this pathway causes decreased lymph gland growth similar to loss of Scalloped and PVF2, demonstrating a requirement for PVR/STAT signaling in the regulation of lymph gland size. Finally, we demonstrate that Scalloped regulates PVR expression and the maintenance of progenitors downstream of PVR/STAT/ADGF signaling. These findings further establish the role of the TEAD family transcription factors in the regulation of important signaling molecules, and expand our mechanistic insight into the balance between progenitor maintenance and proliferation required for the regulation of lymph gland homeostasis.

Molecular characterization and function of a PTEN gene from Litopenaeus vannamei after Vibrio alginolyticus challenge.

PTEN, a tumor suppressor gene, suppresses cell survival, growth, apoptosis, cell migration and DNA damage repair by inhibiting the PI3K/AKT signaling pathway. In this study, the full-length Litopenaeus vannamei PTEN (LvPTEN) cDNA was obtained, containing a 5'UTR of 59bp, an ORF of 1269bp and a 3'UTR of 146bp besides the poly (A) tail. The PTEN gene encoded a protein of 422 amino acids with an estimated molecular mass of 48.3 KDa and a predicted isoelectric point (pI) of 7.6. Subcellular localization analysis revealed that LvPTEN was distributed in both cytoplasm and nucleus, and the tissue distribution patterns showed that LvPTEN was ubiquitously expressed in all the examined tissues. Vibrio alginolyticus challenge induced upregulation of LvPTEN expression. Moreover, RNAi knock-down of LvPTEN in vivo significantly increased the expression of LvAKT mRNA, while reducing that of the downstream apoptosis genes LvP53 and LvCaspase3. LvPTEN knock-down also caused a sharp increase in cumulative mortality, bacterial numbers, and DNA damage in the hemolymph of L. vannamei following V. alginolyticus challenge, together with a sharp decrease in the total hemocyte count (THC). These results suggested that LvPTEN may participate in apoptosis via the PI3K/AKT signaling pathway in L. vannamei, and play an important role in shrimp innate immunity.

Perfil proteico da hemolinfa de Biomphalaria glabrata e linhagens de B. straminea, frente à exposição ao Schistosoma mansoni / Protein profile of hemolymph of Biomphalaria glabrata and strains of B. straminea, against the exposure to the Schistosoma mansoni

O gênero Biomphalaria possui espécies de grande relevância médica uma vez que atuam como hospedeiros intermediários naturais do parasita Schistosoma mansoni, causador da esquistossomose. Dentro desse gênero de moluscos, três espécies são tidas como hospedeiros naturais do parasita, Biomphalaria glabrata, B. straminea e B. tenagophila. O perfil de suscetibilidade à infecção por S. mansoni dentro do gênero é muito variado e muitas pesquisas buscam elucidar a dinâmica da relação parasita-hospedeiro intermediário na finalidade de criar novas medidas de controle da doença. Por isso, esse estudo tem como objetivo determinar o perfil bidimensional de proteínas que podem estar envolvidas na resposta imune contra o S. mansoni comparando duas espécies com diferentes perfis de susceptibilidade B. glabrata, B. straminea além de uma refratária ao S. mansoni, a B. straminea R3. Para isso, os caramujos de cada espécie foram divididos em dois grupos: Infectado, expostos aos miracídios do S. mansoni; e Controle, submetidos ao estresse do processo de infecção livre de miracídios. A hemolinfa foi retirada 24 horas após a exposição. Foi feito o extrato proteico total e determinada a concentração das proteínas totais para cada grupo investigado. As proteínas foram separadas por eletroforese bidimensional onde foi obtido o ponto isoelétrico e peso molecular de todos os spots nos géis...

The Biomphalaria has species of great medical relevance since that act as natural intermediate hosts of the parasite Schistosoma mansoni, which causes schistosomiasis. Within this kind of mollusks, three species are considered natural hosts of the parasite, Biomphalaria glabrata, B. stramineaand B. tenagophila. The profile of usceptibility to S. mansoni infection within the genre is very varied and many studies seek to elucidate the dynamics of host-parasite relationship intermediary in order to create new disease control easures. Therefore, this study aims to determine the two-dimensional profile of proteins that may be involved in the immune response against S. mansonicomparing two species with different susceptibility profiles B. glabrata, B. straminea and a refractory to S. mansoni, B. straminea R3. For that, the snails of each species were divided into two groups: Infected exposed to iracidia of S. mansoni; and control, subjected to stress the miracidia free infection process. The hemolymph was removed 24 hours after exposure. It was made the total protein extract and determined the concentration of total protein for each group investigated. Proteins were separated by two-dimensional electrophoresis was obtained where the isoelectric point and molecular weight of all the spots in the gels...

Effects of waterborne nickel on the physiological and immunological parameters of the Pacific abalone Haliotis discus hannai during thermal stress.

In this study, the 96-h LC50 at 22 and 26 °C values was 28.591 and 11.761 mg/L, respectively, for NiCl2 exposure in the abalone. The alteration of physiological and immune-toxicological parameters such as the total hemocyte count (THC), lysozyme, phenoloxidase (PO), and phagocytosis activity was measured in the abalone exposed to nickel (200 and 400 µg/L) under thermal stress for 96 h. In this study, Mg and THC decreased, while Ca, lysozyme, PO, and phagocytosis activity increased in the hemolymph of Pacific abalone exposed to NiCl2 when compared to a control at both 22 and 26 °C. However, these parameters were not affected by a rise in temperature from 22 to 26 °C in non-exposed groups. Our results showed that NiCl2 below 400 µg/L was able to stimulate immune responses in abalone. However, complex stressors, thermal changes, or NiCl2 can modify the immunological response and lead to changes in the physiology of host-pollutant interactions in the abalone.

Limiting immunopathology: Interaction between carotenoids and enzymatic antioxidant defences.

The release of reactive oxygen and nitrogen species (ROS and RNS) during the inflammatory response generates damages to host tissues, referred to as immunopathology, and is an important factor in ecological immunology. The integrated antioxidant system, comprising endogenous antioxidant enzymes (e.g. superoxide dismutase SOD, and catalase CAT) and dietary antioxidants (e.g. carotenoids), helps to cope with immune-mediated oxidative stress. Crustaceans store large amounts of dietary carotenoids for yet unclear reasons. While being immunostimulants and antioxidants, the interaction of these pigments with antioxidant enzymes remains unclear. Here, we tested the interaction between dietary supplementation with carotenoids and immune challenge on immune defences and the activity of the antioxidant enzymes SOD and CAT, in the amphipod crustacean Gammarus pulex. Dietary supplementation increased the concentrations of circulating carotenoids and haemocytes in the haemolymph, while the immune response induced the consumption of circulating carotenoids and a drop of haemocyte density. Interestingly, supplemented gammarids exhibited down-regulated SOD activity but high CAT activity compared to control ones. Our study reveals specific interactions of dietary carotenoids with endogenous antioxidant enzymes, and further underlines the potential importance of carotenoids in the evolution of immunity and/or of antioxidant mechanisms in crustaceans.

[Cellular immune system of surgical maggots Lucilia sericata (Diptera, Calliphoridae)].

In the hemolymph of surgical maggots Lucilia sericata seven types of hemocytes were revealed. These are prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation. In contrast to Calliphora hyaline cells, this type of hemocytes in cropemptying larvae of Lucilia is elongated or vermiform in shape. Hyaline cells may be transformed to both prothrombocytoids and unstable prophenoloxydase-producing cells. Appearance and differentiation of each hemocyte type is rigidly linked with a definite stage of development. In cellular defense the main role play juvenile plasmatocytes, plasmatocytes II and III and trombocytoides. Juvenile plasmatocytes are the most active ones. After charcoal particles injection they were instantly surrounded by the thick envelope of adhered alien particles and form uniform morules aggregations or conglomerates together with thrombocytoidal agglutinates. Plasmatocytes II and III during the early stages of differentiation may be involved in adhesion and phagocytosis of alien particles and during the last stages in the engulfing of apoptose desintegrated tissues. Thus the cellular defense reaction is assisted by 4 hemocyte types--prophenoloxydase-unstable hyaline cells, thrombocytoids, juvenile plasmatocytes and plasmatocytes I-IV.

[Cellular defense system of some synanthropic dipterans inhabitant of bacterially aggressive environment].

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.

Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria.

Bivalve mollusks of the North Atlantic, most prominently the soft shell clam Mya arenaria, are afflicted with an epidemic transmissible disease of the circulatory system closely resembling leukemia. The disease is characterized by a dramatic expansion of blast-like cells in the hemolymph with high mitotic index. Examination of hemolymph of diseased clams revealed high levels of reverse transcriptase activity, the hallmark of retroviruses and retroelements. By deep sequencing of RNAs from hemolymph, we identified transcripts of a novel retroelement, here named Steamer. The DNA of the element is marked by long terminal repeats and encodes a single large protein with similarity to mammalian retroviral Gag-Pol proteins. Steamer mRNA levels were specifically elevated in diseased hemocytes, and high expression was correlated with disease status. DNA copy number per genome was present at enormously high levels in diseased hemocytes, indicative of extensive reverse transcription and retrotransposition. Steamer activation in M. arenaria is an example of a catastrophic induction of genetic instability that may initiate or advance the course of leukemia.

Effects of repeated hemolymph withdrawals on the hemocyte populations and hematopoiesis in Pomacea canaliculata.

Pomacea canaliculata is a freshwater gastropod considered an invasive pest by several European, North American and Asiatic countries. This snail presents a considerable resistance to pollutants and may successfully face stressful events. Thanks to the unusual possibility to perform several hemolymph collections without affecting its survival, P. canaliculata is a good model to study the hematopoietic process and the hemocyte turnover in molluscs. Here we have analyzed the effects of repeated hemolymph withdrawals on circulating hemocyte populations and pericardial organs, i.e., the heart, the main vessels entering and leaving the heart and the ampulla, of P. canaliculata. Our experiments revealed that the circulating hemocyte populations were maintained constant after 3 collections performed in 48 h. The tissue organization of the heart and the vessels remained unaltered, whereas the ampulla buffered the effects of hemolymph collections acting as hemocyte reservoir, and its original organization was progressively lost by the repeated hemolymph withdrawals. The hematopoietic tissue of P. canaliculata was evidenced here for the first time. It is positioned within the pericardial cavity, in correspondence of the principle veins. Mitoses within the hematopoietic tissue were not influenced by hemolymph collections, and circulating hemocytes never presented mitotic activity.

Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp.

Sulfated galactans (SG) were isolated from the red seaweed Gracilaria fisheri (G. fisheri). Chemical analysis revealed SG contains sulfate (12.7%) and total carbohydrate (42.2%) with an estimated molecular mass of 100 kDa. Structure analysis by NMR and FT-IR spectroscopy revealed that SG is a complex structure with a linear backbone of alternating 3-linked ß-D-galactopyranose and 4-linked 3,6-anhydrogalactose units with partial 6-O-methylate-ß-D-galactopyranose and with sulfation occurring on C4 of D-galactopyranose and C6 of L-galactopyranose units. SG treatment enhanced immune parameters including total haemocytes, phenoloxidase activity, superoxide anions and superoxide dismutase in shrimp Penaeus monodon. Shrimp fed with Artemia salina enriched with SG (100 and 200 µg ml(-1)) and inoculated with white spot syndrome virus (WSSV) showed a significantly lower mortality rate and lower viral VP 28 amplification and expression than control. The results suggest that SG from G. fisheri exhibits immune stimulatory and antiviral activities that could protect P. monodon from WSSV infection.

Blood scent.

Blood cell production is tightly regulated by cell-intrinsic mechanisms and environmental factors. The study by Utpal Banerjee and colleagues and colleagues reveals that, in Drosophila, olfactory signals control hematopoietic progenitor maintenance, thus uncovering a physiological link between sensory perception and hematopoietic response to environmental stress.

Olfactory control of blood progenitor maintenance.

Drosophila hematopoietic progenitor maintenance involves both near neighbor and systemic interactions. This study shows that olfactory receptor neurons (ORNs) function upstream of a small set of neurosecretory cells that express GABA. Upon olfactory stimulation, GABA from these neurosecretory cells is secreted into the circulating hemolymph and binds to metabotropic GABAB receptors expressed on blood progenitors within the hematopoietic organ, the lymph gland. The resulting GABA signal causes high cytosolic Ca(2+), which is necessary and sufficient for progenitor maintenance. Thus, the activation of an odorant receptor is essential for blood progenitor maintenance, and consequently, larvae raised on minimal odor environments fail to sustain a pool of hematopoietic progenitors. This study links sensory perception and the effects of its deprivation on the integrity of the hematopoietic and innate immune systems in Drosophila. PAPERCLIP:

Biomarkers and transcription levels of cancer-related genes in cockles Cerastoderma edule from Galicia (NW Spain) with disseminated neoplasia.

Disseminated neoplasia (DN) is a pathological condition reported for several species of marine bivalves throughout the world, but its aetiology has not yet been satisfactorily explained. It has been suggested that chemical contamination could be a factor contributing to neoplasia. The aim of the present study was to compare cell and tissue biomarkers and the transcription level of cancer-related genes in cockles (Cerastoderma edule) affected by DN with those of healthy cockles in relation to chemical contaminant burdens. For this, cockles were collected from a natural bed in Cambados (Ria de Arousa, Galicia) in May 2009. The prevalence of DN was 12.36% and 3 degrees of DN severity were distinguished. No significant differences in metal accumulation, non-specific inflammatory responses and parasites were observed between healthy and DN-affected cockles. Lysosomal membrane stability was significantly reduced in cockles affected by DN, which indicates a poorer health condition. Very low frequencies of micronuclei were recorded and no significant differences were detected between DN severity groups. Haemolymph analyses showed a higher frequency of mitotic figures and binucleated cells in cockles affected by moderate and heavy DN than in healthy ones. Neoplastic animals showed significantly higher transcription levels of p53 and ras than healthy cockles and mutational alterations in ras gene sequence were detected. Low concentrations of metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters were measured in cockles from Cambados. In conclusion, cockles affected by DN suffer a general stress situation and have altered patterns of cancer-related gene transcription. Further studies are in progress to elucidate mechanisms of carcinogenesis in this species.

Phagocytic activity of Limulus polyphemus amebocytes in vitro.

Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlantic horseshoe crab, Limulus polyphemus, possesses a single immune cell type, the granular amebocyte. Amebocytes release a repertoire of potent immune effectors in the presence of pathogens, and function in hemostasis. In contrast to other arthropod immunocytes, the properties of amebocyte phagocytosis remain poorly characterised, restricted by the technical challenges associated with handling these labile cells. We have addressed these challenges and observed the internalisation of microbial and synthetic targets by amebocytes in vitro. Confirmation of target internalisation was achieved using a combination of fluorescent quenching and lipophilic membrane probes: R18 and FM 1-43. Viability, morphological integrity and functionality of extracted amebocytes appeared to be retained in vitro. The phagocytic properties of L. polyphemus amebocytes described here, in the absence of endotoxin, are similar to those observed for arthropod immunocytes and mammalian neutrophils.