Pseudomonas boanensis sp. nov., a bacterium isolated from river water used for household purposes in Boane District, Mozambique.

A Gram-negative rod with a single polar flagellum was isolated from a freshwater reservoir used for household purposes in Boane District, near Maputo, Mozambique, and designated as strain DB1T. Growth was observed at 30-42 °C (optimum, 30-37 °C) and with 0.5-1.5 % NaCl. Whole-genome-, rpoD- and 16S rRNA-based phylogenies revealed this isolate to be distant from other Pseudomonas species with Pseudomonas resinovorans, Pseudomonas furukawaii and Pseudomonas lalkuanensis being the closest relatives. Phenotypic analyses of strain DB1T showed marked differences with respect to type strains P. resinovorans CCUG 2473T, P. lalkuanensis CCUG 73691T, P. furukawaii CCUG 75672T and Pseudomonas otiditis CCUG 55592T. Taken together, our results indicate that strain DB1T is a representative of a novel species within the genus Pseudomonas for which the name Pseudomonas boanensis is proposed. The type strain is DB1T (=CCUG 62977T=CECT 30359T).

Alteration of host-pathogen interactions in the wake of climate change - Increasing risk for shellfish associated infections?

The potential for climate-related spread of infectious diseases through marine systems has been highlighted in several reports. With this review we want to draw attention to less recognized mechanisms behind vector-borne transmission pathways to humans. We have focused on how the immune systems of edible marine shellfish, the blue mussels and Norway lobsters, are affected by climate related environmental stressors. Future ocean acidification (OA) and warming due to climate change constitute a gradually increasing persistent stress with negative trade-off for many organisms. In addition, the stress of recurrent hypoxia, inducing high levels of bioavailable manganese (Mn) is likely to increase in line with climate change. We summarized that OA, hypoxia and elevated levels of Mn did have an overall negative effect on immunity, in some cases also with synergistic effects. On the other hand, moderate increase in temperature seems to have a stimulating effect on antimicrobial activity and may in a future warming scenario counteract the negative effects. However, rising sea surface temperature and climate events causing high land run-off promote the abundance of naturally occurring pathogenic Vibrio and will in addition, bring enteric pathogens which are circulating in society into coastal waters. Moreover, the observed impairments of the immune defense enhance the persistence and occurrence of pathogens in shellfish. This may increase the risk for direct transmission of pathogens to consumers. It is thus essential that in the wake of climate change, sanitary control of coastal waters and seafood must recognize and adapt to the expected alteration of host-pathogen interactions.

Ocean acidification and host-pathogen interactions: blue mussels, Mytilus edulis, encountering Vibrio tubiashii.

Ocean acidification (OA) can shift the ecological balance between interacting organisms. In this study, we have used a model system to illustrate the interaction between a calcifying host organism, the blue mussel Mytilus edulis and a common bivalve bacterial pathogen, Vibrio tubiashii, with organisms being exposed to a level of acidification projected to occur by the end of the 21st century. OA exposures of the mussels were carried out in relative long-term (4 months) and short-term (4 days) experiments. We found no effect of OA on the culturability of V. tubiashii, in broth or in seawater. OA inhibited mussel shell growth and impaired crystalline shell structures but did not appear to affect mussel immune parameters (i.e haemocyte counts and phagocytotic capacity). Despite no evident impact on host immunity or growth and virulence of the pathogen, V. tubiashii was clearly more successful in infecting mussels exposed to long-term OA compared to those maintained under ambient conditions. Moreover, OA exposed V. tubiashii increased their viability when exposed to haemocytes of OA-treated mussel. Our findings suggest that even though host organisms may have the capacity to cope with periods of OA, these conditions may alter the outcome of host-pathogen interactions, favouring the success of the latter.

Manganese as a Possible Anticancer Enhancer in Docetaxel Treatment of Prostate Cancer Cells.

BACKGROUND/AIM: Treatment of castration-resistant prostate cancer with docetaxel (DOC) often leads to resistance. In this study, we investigated whether manganese (Mn) has the potential to enhance treatment when combined with DOC. MATERIALS AND METHODS: PC3 cells were exposed to DOC or Mn individually and in combination and cell viability was analysed in a dose- and time-dependent manner. Cell toxicity, cell cycle analysis and apoptotic protein levels were determined after 48 h of treatment. RESULTS: Mn in combination with different concentrations of DOC significantly enhanced the inhibitory effect on cell viability. Although the lowest dose did not cause mitotic arrest, DOC increased toxicity, which was reduced when combined with Mn. Protein analyses indicated that Mn compensates for the suppression of death receptors when combined with a low concentration of DOC and induced non-apoptotic pathways when combined with a higher concentration. CONCLUSION: Combining DOC and Mn may allow for a reduction in DOC concentration with the potential to reduce side effects.

Stress biology and immunology in Nephrops norvegicus.

The Norway lobster Nephrops norvegicus lives at low-light depths, in muddy substrata of high organic content where water salinities are high and fluctuations in temperature are moderate. In this environment, the lobsters are naturally exposed to a number of potential stressors, many of them as a result of the surficial breakdown of organic material in the sediment. This process (early diagenesis) creates a heterogeneous environment with temporal and spatial fluctuations in a number of compounds such as oxygen, ammonia, metals, and hydrogen sulphide. In addition to this, there are anthropogenically generated stressors, such as human-induced climate change (resulting in elevated temperature and ocean acidification), pollution and fishing. The lobsters are thus exposed to several stressors, which are strongly linked to the habitat in which the animals live. Here, the capacity of Nephrops to deal with these stressors is summarised. Eutrophication-induced hypoxia and subsequent metal remobilisation from the sediment is a well-documented effect found in some wild Nephrops populations. Compared to many other crustacean species, Nephrops is well adapted to tolerate periods of hypoxia, but prolonged or severe hypoxia, beyond their tolerance level, is common in some areas. When the oxygen concentration in the environment decreases, the bioavailability of redox-sensitive metals such as manganese increases. Manganese is an essential metal, which, taken up in excess, has a toxic effect on several internal systems such as chemosensitivity, nerve transmission and immune defence. Since sediment contains high concentrations of metals in comparison to sea water, lobsters may accumulate both essential and non-essential metals. Different metals have different target tissues, though the hepatopancreas, in general, accumulates high concentrations of most metals. The future scenario of increasing anthropogenic influences on Nephrops habitats may have adverse effects on the fitness of the animals.

Simulated climate change causes immune suppression and protein damage in the crustacean Nephrops norvegicus.

Rising atmospheric carbon dioxide concentration is causing global warming, which affects oceans by elevating water temperature and reducing pH. Crustaceans have been considered tolerant to ocean acidification because of their retained capacity to calcify during subnormal pH. However, we report here that significant immune suppression of the Norway lobster, Nephrops norvegicus, occurs after a 4-month exposure to ocean acidification (OA) at a level predicted for the year 2100 (hypercapnic seawater with a pH lowered by 0.4 units). Experiments carried out at different temperatures (5, 10, 12, 14, 16, and 18°C) demonstrated that the temperature within this range alone did not affect lobster immune responses. In the OA-treatment, hemocyte numbers were reduced by almost 50% and the phagocytic capacity of the remaining hemocytes was inhibited by 60%. The reduction in hemocyte numbers was not due to increased apoptosis in hematopoetic tissue. Cellular responses to stress were investigated through evaluating advanced glycation end products (AGE) and lipid oxidation in lobster hepatopancreata, and OA-treatment was shown to significantly increase AGEs', indicating stress-induced protein alterations. Furthermore, the extracellular pH of lobster hemolymph was reduced by approximately 0.2 units in the OA-treatment group, indicating either limited pH compensation or buffering capacity. The negative effects of OA-treatment on the nephropidae immune response and tissue homeostasis were more pronounced at higher temperatures (12-18°C versus 5°C), which may potentially affect disease severity and spread. Our results signify that ocean acidification may have adverse effects on the physiology of lobsters, which previously had been overlooked in studies of basic parameters such as lobster growth or calcification.

Possibility of mixed progenitor cells in sea star arm regeneration.

In contrast to most vertebrates, invertebrate deuterostome echinoderms, such as the sea star Asterias rubens, undergo regeneration of lost body parts. The current hypothesis suggests that differentiated cells are the main source for regenerating arm in sea stars, but there is little information regarding the origin and identity of these cells. Here, we show that several organs distant to the regenerating arm responded by proliferation, most significantly in the coelomic epithelium and larger cells of the pyloric caeca. Analyzing markers for proliferating cells and parameters indicating cell ageing, such as levels of DNA damage, pigment, and lipofuscin contents as well as telomere length and telomerase activity, we suggest that cells contributing to the new arm likely originate from progenitors rather than differentiated cells. This is the first study showing that cells of mixed origin may be recruited from more distant sources of stem/progenitor cells in a sea star, and the first described indication of a role for pyloric caeca in arm regeneration. Data on growth rate during arm regeneration further indicate that regeneration is at the expense of whole animal growth. We propose a new working hypothesis for arm regeneration in sea stars involving four phases: wound healing by coelomocytes, migration of distant progenitor cells of mixed origin including from pyloric caeca, proliferation in these organs to compensate for cell loss, and finally, local proliferation in the regenerating arm.

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L).

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens', was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.

Immunosuppression of aquatic organisms exposed to elevated levels of manganese: From global to molecular perspective.

Manganese (Mn) is an essential trace metal for all organisms. However, in excess it causes toxic effects but the impact on aquatic environments has so far been highly overlooked. Manganese is abundant both in costal and deep sea sediments and becomes bioavailable (Mn2+) during redox conditions. This is an increasing phenomenon due to eutrophication-induced hypoxia and aggravated through the ongoing climate change. Intracellular accumulation of Mn2+ causes oxidative stress and activates evolutionary conserved pathways inducing apoptosis and cell cycle arrest. Here, studies are compiled on how excess of dissolved Mn suppresses the immune system of various aquatic organisms by adversely affecting both renewal of immunocytes and their functionality, such as phagocytosis and activation of pro-phenoloxidase. These impairments decrease the animal's bacteriostatic capacity, indicating higher susceptibility to infections. Increased distribution of pathogens, which is believed to accompany climate change, requires preserved immune sentinel functions and Mn can be crucial for the outcome of host-pathogen interactions.

A comparative study on the influence of manganese on the bactericidal response of marine invertebrates.

Manganese, Mn, is a naturally abundant metal in marine sediments. During hypoxic conditions the metal converts into a bioavailable state and can reach levels that have been shown immunotoxic to the crustacean Nephrops norvegicus. For this species it has previously been shown that exposure to 15 mg L(-1) of Mn decreased the number of circulating haemocytes while it for the echinoderm Asterias rubens increased the number of coelomocytes. Here, we compared if five days of exposure to the same concentration of Mn affects the bactericidal capacity of these two species and the mollusc Mytilus edulis when inoculated with the bacterium Vibrio parahaemolyticus. Viable counts of the bacteria were investigated at a time-course post-injection in the blood and the digestive glands of Mn-exposed and un-exposed (controls) animals. Accumulation of Mn was also analyzed in these tissues. When exposed to Mn the haemocyte numbers were significantly reduced in M. edulis and it was shown that the bactericidal capacity was impaired in the mussels as well as in N. norvegicus. This was most obvious in the digestive glands. These two species also showed the highest accumulation of the metal. In A. rubens the bactericidal capacity was not affected and the metal concentration was similar to the exposure concentration. After a recovery period of three days the concentration of Mn was significantly reduced in all three species. However, in M. edulis and N. norvegicus it was still double that of A. rubens which could explain the remaining bactericidal suppression observed in N. norvegicus. This study pointed out that exposure to such Mn-levels that are realistic to find in nature could have effects on the whole organism level, in terms of susceptibility to infections. The effect seemed associated to the accumulated concentration of Mn which differed on species level.

Tracing of norovirus outbreak strains in mussels collected near sewage effluents.

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia.

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.

Manganese effects on haematopoietic cells and circulating coelomocytes of Asterias rubens (Linnaeus).

Manganese (Mn) is a naturally abundant metal in marine sediments where it mainly occurs as MnO(2). During hypoxic conditions it is converted into a bioavailable state, Mn(2+), and can reach levels that previously have shown effects on immune competent cells of the crustacean, Nephrops norvegicus. Here we investigated if Mn also affects circulating coelomocytes and their renewal in the common sea star, Asterias rubens, when exposed to concentrations of Mn that can be found in nature. When the sea stars were exposed to Mn it accumulated in the coelomic fluid and the number of circulating coelomocytes, in contrast to what was recorded in Nephrops, increased significantly. By using the substitute nucleotide, 5-bromo-2'-deoxyuridine, BrdU, for tracing cell division and by recording mitotic index by nuclei staining, we found that Mn induced proliferation of cells from a putative haematopoietic tissue, the coelomic epithelium. In addition, the haematopoietic tissue and coelomocytes showed stress response in terms of changes in HSP70 levels and protein carbonyls, as judged by immunohistochemistry and Western blotting. Measurement of dehydrogenase activity, using MTS/PMS, revealed that Mn showed cytotoxic properties. We also found that the phagocytotic capacity of coelomocytes was significantly inhibited by Mn. It was concluded that the exposure of A. rubens to Mn induced renewal of coelomocytes and impaired their immune response.

Manganese Inhibits Viability of Prostate Cancer Cells.

BACKGROUND/AIM: Androgen deprivation therapy is usually in the initial phase a successful treatment for prostate cancer but eventually most patients develop androgen-independent metastatic disease. This study investigated if manganese (Mn) reduces viability of prostate cancer via induction of apoptosis. MATERIALS AND METHODS: The prostate cancer cell lines PC3, DU145 and LNCaP underwent dose- and time-dependent screening of viability, analyzed by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Flow cytometry was used for the cell-cycle and apoptosis analyses. Intracellular Mn concentration was measured using inductively coupled plasma-mass spectrometry. RESULTS: At Mn concentrations of 200-1000 µM, the effect on viability was most pronounced in PC3 followed by LNCaP cells. These cell lines also showed higher intracellular concentration of Mn compared to DU145. In all cell lines, Mn increased the proportion of cells arrested in the G0/G1 phase and induced apoptosis. CONCLUSION: To our knowledge, this is the first report demonstrating Mn as a potential agent in prostate cancer therapy.

The persistence of infectious adenovirus (type 35) in mussels (Mytilus edulis) and oysters (Ostrea edulis).

The aim of this study was to provide information for improving risk assessment of viral contaminants in bivalves. The persistence of viable adenovirus type 35 (Ad35) after controlled contaminations of blue mussels, Mytilus edulis, and oysters, Ostrea edulis, was studied. Bivalves, kept in running seawater at two different temperatures (4 and 18 degrees C) were sampled after 1, 3, 7, 14, 21, 35, 42, 49, 56, and 70 days. Virus particles were separated from the gills and the digestive gland through ultra high-speed centrifugation. Qualitative PCR analyses of DNA in the virus extracts showed that Ad35 was detectable for 6-10 weeks and quantitative real-time PCR verified a gradual but not linear decrease in copy numbers, within this time interval. The virus genome was detectable to the same degree on the gills as in the digestive gland. When viral extractions were inoculated on A549 cells to investigate the cytopathic effect (CPE) it was shown that Ad35 stayed infectious in oysters, kept at 4 degrees C, for about six weeks, which was double the time compared to that for mussels. The detection of the viral genome exceeded the persistence of their infectivity, in most cases with 4-6 weeks. The data were highly variable and the sporadic occurrence of high numbers of accumulated viruses and their remaining infectivity is seemingly a significant factor regarding food safety.

Manganese induced apoptosis in haematopoietic cells of Nephrops norvegicus (L.).

Manganese (Mn) is highly abundant as MnO2 in marine sediments. During hypoxia in bottom waters, the reduced bioavailable fraction of manganese, Mn2+, increases. Thereby, Norway lobster, Nephrops norvegicus, can experience concentrations up to 1000 times normoxic levels. A previous study has shown that exposure to a realistic concentration of 20 mg l(-1) of Mn for 10 days reduced the number of circulating haemocytes in N. norvegicus significantly. Here we aimed to investigate if apoptosis contributes to the Mn-induced haemocytopenia, with the overall hypothesis that Mn induces apoptosis in a time and concentration dependent manner. N. norvegicus were exposed to Mn (0, 5, 10 and 20 mg l(-1)) for 5 and 10 days. After 5 days of exposure the total haemocyte counts were not affected. However, after 10 days there was a gradual decrease in cell numbers, reaching a reduction by 44% when the animals were exposed to 20 mg Mn l(-1). Apoptosis in cells, released from the haematopoietic tissue, was investigated by using TUNEL assay, which detects specific DNA strand breaks. The fraction of apoptotic cells gradually increased from 2.5% in un-exposed lobsters to 15% in those exposed to 20 mg l(-1) but there was no difference related to the exposure time. A gradual increase of apoptosis was further confirmed by electrophoretic DNA-ladder formation, however to a lower extent in lobsters exposed during 5 days. Cell viability, determined by metabolic activity and cell membrane integrity, was not reduced, indicating that apoptosis rather than necrosis caused reduced number of haemocytes. It was concluded that apoptosis seemed to increase already after 5 days of 5 mg l(-1) of Mn-exposure, although exposure for 10 days was required before it was reflected in the haemocyte numbers. Reduced numbers of haemocytes may increase the prevalence for infections in N. norvegicus in their natural habitat.

Complete Genome Sequences of Two Marine Vibrio cholerae Strains Isolated from the South Coast of Sweden.

Vibrio cholerae serogroups O1 and O139 are commonly associated with diarrhea, while non-O1-O139 strains may cause wound infections. Here, we present the genome sequences of two V. cholerae strains isolated from blue mussels (Mytilus edulis) collected in coastal waters of southern Sweden.

Shellfish and public health: a Swedish perspective.

Bivalves are ancient animals that feed by filtering large volumes of water. In this way, phytoplankton, bacteria and viruses from the water column are greatly concentrated in the mussels. The hazards associated with the consumption of mussels are thus dependent on the occurrence and composition of toxic algae and human microbial pathogens in the areas where shellfish are grown. Diarrheic shellfish toxins have occurred regularly in Sweden during the past 27 years. Peaks of toxins in mussels are mostly recorded from October to December, but the pattern can differ significantly due to location and year, making it hard to predict toxin levels in mussels. With an expansion of aquaculture and a subsequent increase in seafood consumption, better risk management is needed to minimize the effects on humans of algal toxins and human pathogens. New control strategies that have to be implemented are: i) proper site selection of culture installations; ii) regular and cost-efficient monitoring of algae, bacteria and viruses; iii) new indicators for fecal contamination, suitable for the specific locations where shellfish grow; iv) rapid dissemination of information to the industry and public, including risk assessment and advice on how to cope with the situation.

Improving marine water quality by mussel farming: a profitable solution for Swedish society.

Eutrophication of coastal waters is a serious environmental problem with high costs for society globally. In eastern Skagerrak, reductions in eutrophication are planned through reduction of nitrogen inputs, but it is unclear how this can be achieved. One possible method is the cultivation of filter-feeding organisms, such as blue mussels, which remove nitrogen while generating seafood, fodder and agricultural fertilizer, thus recycling nutrients from sea to land. The expected effect of mussel farming on nitrogen cycling was modeled for the Gullmar Fjord on the Swedish west coast and it is shown that the net transport of nitrogen (sum of dissolved and particulate) at the fjord mouth was reduced by 20%. Existing commercial mussel farms already perform this service for free, but the benefits to society could be far greater. We suggest that rather than paying mussel farmers for their work that nutrient trading systems are introduced to improve coastal waters. In this context an alternative to nitrogen reduction in the sewage treatment plant in Lysekil community through mussel farming is presented. Accumulation of bio-toxins has been identified as the largest impediment to further expansion of commercial mussel farming in Sweden, but the problem seems to be manageable through new techniques and management strategies. On the basis of existing and potential regulations and payments, possible win-win solutions are suggested.

Bacteriostatic suppression in Norway lobster (Nephrops norvegicus) exposed to manganese or hypoxia under pressure of ocean acidification.

Future ocean acidification (OA) and warming following climate change elicit pervasive stressors to the inhabitants of the sea. Previous experimental exposure to OA for 16 weeks at pH levels predicted for 2100 has shown to result in serious immune suppression of the Norway lobster, Nephrops norvegicus. The lobsters are currently affected by stressors such as periodical hypoxia inducing high levels of bioavailable manganese (Mn). Here, we aimed to investigate possible effects of interactions between OA and these stressors on total hemocyte counts (THCs) and on recovery of inoculated bacteria in the lobsters, measured as a proxy for bacteriostatic response. The effects were judged by following numbers of culturable Vibrio parahaemolyticus in hepatopancreas, 4 and 24h post inoculation in lobsters kept in replicate tanks with six different treatments: either ambient (pCO2∼500 µatm/pH∼8.1 U) or CO2-manipulated seawater (OA; pCO2∼1550 µatm/pH∼7.6 U) for 8 weeks. During the last 2 weeks, additional stress of either hypoxia (∼23% oxygen saturation) or Mn (∼9 mg L(-1)) was added except in control treatments. Our results showed clear effect on bacteriostatic response in Norway lobsters exposed to these stressors. In lobsters kept in ambient seawater without additional stressors, the number of culturable bacteria in hepatopancreas was reduced by ∼34%. In combined treatment of ambient seawater and hypoxia, the reduction was ∼23%, while in the Mn-exposed animals, there was no reduction at all. This was also the case in all OA treatments where mean numbers of culturable V. parahaemolyticus tended to increase. In lobsters from ambient seawater with or without hypoxia, the THC was not significantly different as was also the case in OA without additional stressors. However, in OA treatments combined with either hypoxia or Mn, THC was reduced by ∼35%. While the reduction of culturable V. parahaemolyticus in lobsters was clearly affected by these stressors, we found no notable effects on growth, survival or hemolytic properties of the bacteria itself. Thus, we conclude that this predicted stress scenario is beneficial for the pathogen in its interaction with the host. As OA proceeds, it may force the health of the ecologically and economically important N. norvegicus to a tipping point if exposed to more short-term stressors such as the periodical events of hypoxia and Mn. This could impact lobster condition and biomass and may as well increase the risk for bacterial transmission to consumers.