Manganese (Mn) is one of the most abundant elements on earth, particularly in the soft bottom sediments of the oceans. As a micronutrient Mn is essential in the metabolic processes of organisms. However, at high concentrations the metal becomes a neurotoxin with well-documented effects. As a consequence of euthrophication, manganese is released from bottom sediments of coastal areas and the Norway lobsters, Nephrops norvegicus, can experience high levels of bioavailable Mn(2+). Here, we present the first report showing that Mn also affects several fundamental processes in the mobilisation and activation of immunoactive haemocytes. When N. norvegicus was exposed to a realistic [Mn(2+)] of 20 mg l(-1) for 10 days 24.1 microg ml(-1) was recorded in the haemolymph. At this concentration the total haemocyte count was reduced by ca. 60%. By using BrdU as a tracer for cell division, it was shown that the proliferation rate in the haematopoietic tissue did not increase, despite the haemocytepenia. A gene coding for a Runt-domain protein, known to be involved in maturation of immune active haemocytes in a variety of organisms, was identified also in haemocytes of N. norvegicus. The expression of this gene was >40% lower in the Mn-exposed lobsters as judged by using a cDNA probe and the in situ hybridisation technique. In response to non-self molecules, like lipopolysaccharide (LPS), the granular haemocytes of arthropods are known to degranulate and thereby release and activate the prophenoloxidase system, necessary for their immune defence. A degranulation assay, tested on isolated granular haemocytes, showed about 75% lower activity in the Mn-exposed lobsters than that for the unexposed. Furthermore, using an enzymatic assay, the activation per se of prophenoloxidase by LPS was found blocked in the Mn-exposed lobsters. Taken together, these results show that Mn exposure suppressed fundamental immune mechanisms of Norway lobsters. This identifies a potential harm that also exists for other organisms and should be considered when increasing the distribution of bioavailable Mn, as has been done through recently introduced applications of the metal.

We compared effects of exposure to predict near-future (2100) ocean acidification (OA; pH 7.7) and normal seawater (Control; pH 8.1) on immune and stress responses in the adult sea star Asterias rubens. Analyses were made after one week and after six months of continuous exposure. Following one week exposure to acidified water, the pH of coelomic fluid was significantly reduced. Levels of the chaperon Hsp70 were elevated while key cellular players in immunity, coelomocytes. were reduced by approximately 50%. Following long-term exposure (six months) levels of Hsp70 returned to control values, whereas immunity was further impaired, evidenced by the reduced phagocytic capacity of coelomocytes and inhibited activation of p38 MAP-kinase. Such impacts of reduced seawater pH may have serious consequences for resistance to pathogens in a future acidified ocean.

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 (THC) 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 24 h post inoculation in lobsters kept in replicate tanks with six different treatments; either ambient (pCO2∼ 500 μatm/pH∼8.1 units) or CO2 manipulated seawater (OA; pCO2 1550 μatm/pH∼7.6 units) for 8 weeks. During the last two weeks additional stress of either hypoxia (∼23% oxygen saturation) or Mn (∼9 mgL−1) was added except in control treatments. Our results showed clear effect on bacteriostatic response in Norway lobsters exposed to these stressors. In lobster 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 total hemocyte count (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.

In the oceans, naturally occurring manganese (Mn) is released from the sediments during events of hypoxia. While neuro- and immuno-toxic effects of bioavailable manganese are well documented for crustaceans, studies of similar effects of manganese on other marine invertebrates are comparatively few. Here, we developed a new functional test "the repeated turning assay" to investigate if manganese exposure at ∼12mgL(-1) affected motoric behaviour of two asteroid echinoderms, the Common sea star, Asterias rubens, and the Black brittle star, Ophiocomina nigra. By measuring of the turning-over capacity, from dorsal to ventral position, after one and two weeks of manganese exposure, we showed that for both species Mn exposure significantly delayed the ability to turn. After a recovery period of two weeks, the capacity of turning-over was not restored to that of unexposed animals neither for A. rubens nor for O. nigra. Further investigation of sea stars showed that Mn accumulated ∼5 fold in the tube feet, organs involved in their turning-over activity, and the high concentration remained after the recovery period. In the tube feet we also recorded an increased activity of acetylcholinesterase (AChE), here used as a proxy for neuromuscular disturbances. The results indicated that Mn induces neuromuscular disturbance in echinoderms which is important news, given that previous studies have concluded that adult echinoderms are relatively tolerant to Mn.

Manganese (Mn) is a naturally abundant metal and particularly so in soft-bottom oceanic sediments where it generally occurs bound in a four-valent colloidal state as MnO2. When hypoxic conditions occur in bottom waters, the metal reduces to the bioavailable ion Mn2+ and can reach concentrations known to have immunotoxic effects in the crustacean Nephrops norvegicus, reducing numbers of circulating haemocytes as a consequence. However, we have previously shown that Mn seems to have a contrasting effect on the echinoderm Asterias rubens in which it triggers the proliferation of haematopoietic cells and increases coelomocyte numbers. Since elevated Mn levels mostly co-occur with hypoxia in nature, here we investigated whether hypoxia has a negative effect on haematopoiesis. Proliferation and differentiation of coelomocytes and cells in the coelomic epithelium of A. rubens were compared after 3 days of exposure to realistic levels of Mn, hypoxia or a combination of these two parameters. We can confirm that Mn elevated numbers of coelomocytes and increased proliferation of epithelial cells, but hypoxia did not affect these levels. However, hypoxia did affect differentiation of these cells as judged by investigating the expression of a Runt domain transcription factor, which was also cloned and sequenced. Through comparative quantification using a real time PCR technique, we found that exposure to hypoxia had a clearly stimulating effect on mRNA expression of Runt gene in both coelomocytes and epithelial cells. These results indicate that during hypoxic conditions the composition of coelomocyte sub-populations changed.

A laboratory study on the sublethal effects of malathion on the net-spinning behavior of the caddisfly larvae Hydropsycheslossonae was conducted in order to assess the potential of net anomalies as an indicator of chronic exposure to organophosphorus insecticides. Two anomalies were identified after chronic exposure to 0.01, 0.05, 0.1, 0.5 and 1.0 μg l⁻¹ malathion. The first was a distortion of the midline meshes where the normal diamond shape structure was disrupted and the meshes were separated by extra strands (called ‘midline’ anomaly). The second aberration observed was a significant decrease in net symmetry. Both anomalies were highly correlated to the toxic action of malathion, i.e. inhibition of the acetylcholinesterase enzyme (AChE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of capture nets did not show any modification of silk polypeptides after exposure to malathion, indicating that net distortions were not related to silk composition. Both anomalies seem to represent the symptoms of the specific toxic action of malathion; nevertheless, they can serve as an index of the physiological condition of the larvae, especially the midline anomaly. The symmetry of the nets decreased significantly after exposure to 0.5 and 1.0 μg l⁻¹. However, the toxicity curves (EC₅₀) showed that the sensitivity threshold for the midline anomaly ranged from 0.11 to 0.28 μg l⁻¹, which reflect more realistic exposure to concentrations expected to occur in the field. Hence, the use of capture net anomalies of hydropsychid larvae could represent a valuable indicator of sublethal toxicity induced by malathion and other organophosphorus insecticides in running waters.