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.

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.

Effects of depth and overgrowth of ephemeral macroalgae on a remote subtidal NE Atlantic eelgrass (Zostera marina) community.

We conducted a short-term field sampling complemented with time integrating stable isotope analysis to holistically investigate status and ecological interactions in a remote NE Atlantic Zostera marina meadow. We found high nutrient water concentrations, large biomass of fast-growing, ephemeral macroalgae, low abundance, and biodiversity of epifauna and a food web with thornback ray (Raja clavata) as intermediate and cod (Gadus morhua) as top predator. We observed no variation with increasing depth (3.5-11 m) except for decreasing shoot density and biomass of Zostera and macroalgae. Our results indicate that the Finnøya Zostera ecosystem is eutrophicated. During the past three to four decades, nutrients from aquaculture have steadily increased to reach 75% of anthropogenic input while the coastal top predator cod has decreased by 50%. We conclude that bottom-up regulation is a predominant driver of change since top-down regulation is generally weak in low density and exposed Zostera ecosystems such as Finnøya.

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.

Severe shifts of Zostera marina epifauna: Comparative study between 1997 and 2018 on the Swedish Skagerrak coast.

The interaction between bottom-up and top-down processes in coastal ecosystems has been scarcely studied so far. Temporal changes in trophic interactions of Zostera marina along the Swedish west coast are relatively well studied, with the exception of epifaunal communities. Epifauna was used as a model study to explore resource (bottom-up) or predator (top-down) regulated in a vegetated ecosystem. We conducted a 21-year comparative study (1997 and 2018) using epifauna of 19 Zostera marina meadows along the Swedish Skagerrak coast. Large changes were observed in the composition of small (0.2-1 mm) and large (>1 mm) epifauna. In the small-sized epifauna, the nematode Southernia zosterae and harpacticoids showed an increase of 90% and a decrease of 50% of their abundances, respectively. In the large-sized epifauna, the polychaete Platynereis dumerilii and chironomid larvae were absent in 1997 but thrived in 2018 (>2000 ind. m-2). Mesoherbivores (Idoteids and gammarids) were locally very abundant in 1997 but disappeared in 2018. An 83% decline of mytilids settling in Zostera marina leaves was observed. Our results showed that epifauna is predominantly top-down regulated. An integrative framework of the study area is outlined to shed light on the causes and consequences of the environmental shifts reported in Zostera meadows from the northern Skagerrak area throughout the last three decades.

Dynamics of seagrass meadows on the Swedish Skagerrak coast.

Seagrasses have declined in many places around the world, and the Swedish Skagerrak coast is no exception. Between the 1980s and 2000, the cover of eelgrass (Zostera marina L.) on the Swedish Skagerrak coast decreased about 60%. In the present study, the sites that were investigated in the 1980s and 2000 were revisited (1655 ha) in 2003 and 2004 to investigate long and short term temporal and spatial differences in eelgrass coverage. The distribution of eelgrass was mapped from a small boat using an aquascope and a GPS receiver. No variation in total (about 150 km) or regional (about 20 km) eelgrass cover was found between 2000 and 2004, but there were substantial changes (both losses and gains) in meadow size on a local (less than about 2 km) scale. The results have implications for management of shallow soft bottoms, particularly because it was shown that even though no eelgrass is found in a single survey in a specific area, there could be eelgrass growing there in the following years.

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.

Eelgrass (Zostera marina) Food Web Structure in Different Environmental Settings.

This study compares the structure of eelgrass (Zostera marina L.) meadows and associated food webs in two eelgrass habitats in Denmark, differing in exposure, connection to the open sea, nutrient enrichment and water transparency. Meadow structure strongly reflected the environmental conditions in each habitat. The eutrophicated, protected site had higher biomass of filamentous algae, lower eelgrass biomass and shoot density, longer and narrower leaves, and higher above to below ground biomass ratio compared to the less nutrient-enriched and more exposed site. The faunal community composition and food web structure also differed markedly between sites with the eutrophicated, enclosed site having higher biomass of consumers and less complex food web. These relationships resulted in a column shaped biomass distribution of the consumers at the eutrophicated site whereas the less nutrient-rich site showed a pyramidal biomass distribution of consumers coupled with a more diverse consumer community. The differences in meadow and food web structure of the two seagrass habitats, suggest how physical setting may shape ecosystem response and resilience to anthropogenic pressure. We encourage larger, replicated studies to further disentangle the effects of different environmental variables on seagrass food web structure.

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.

Motoric impairment following manganese exposure in asteroid echinoderms.

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 ∼12 mg L(-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 induced immune suppression of the lobster, Nephrops norvegicus.

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.

Manganese accumulation by the antennule of the Norway lobster Nephrops norvegicus (L.) as a biomarker of hypoxic events.

In laboratory tests, manganese accumulation by the appendages of the sediment burrowing Norway lobster. Nephrops norvegicus (L.) (including the lateral antennules) was approximately three times greater [600 microg Mn g(-1) (dry weight) after 5 days in 20 mg Mn l(-1)] than that by the carapace. The accumulation was linearly dose-dependent (10-40 mg Mn l(-1)) and duration-dependent (2-30 days). and showed no decrease after 3 weeks in undosed seawater. A high manganese uptakc to the lateral antennules during hypoxia in the field was verified from the SE Kattegat, Sweden. These results indicate that accumulation of Mn on the mobile appendages of the Norway lobster fulfils most of the criteria for a biomarker of exposure to hypoxia. Using these measurements in conjunction with Mn concentrations in the internal tissues, it may be possible to resolve both the timing and the extent of the Mn exposure and the underlying hypoxic event.

Vanishing seagrass (Zostera marina, L.) in Swedish coastal waters.

Along the Swedish Skagerrak coast eelgrass (Zostera marina) is a dominant phanerogam on shallow soft bottoms. Eelgrass meadows are important biotopes for many crustacean and fish species being either migratory or stationary. During the 1980s, inventories of the shallow coastal areas with eelgrass have been carried out along the Swedish west coast as a basis for coastal zone management. In the present study we revisited 2000 ha of eelgrass meadows in 5 coastal regions along 200 km of the Skagerrak coast. The inventory was made with the same methods (aquascope) as during the 1980s, but increasing the mapping accuracy by using a Global Positioning System (GPS). The results from this study show that the areal extension of Zostera marina has decreased 58% in 10-15 years with great regional variations. The decline was mainly restricted to the shallow parts of the meadow. The causes and ecological consequences are discussed.

Distribution, structure and function of Nordic eelgrass (Zostera marina) ecosystems: implications for coastal management and conservation.

This paper focuses on the marine foundation eelgrass species, Zostera marina, along a gradient from the northern Baltic Sea to the north-east Atlantic. This vast region supports a minimum of 1480 km2 eelgrass (maximum >2100 km2), which corresponds to more than four times the previously quantified area of eelgrass in Western Europe.Eelgrass meadows in the low salinity Baltic Sea support the highest diversity (4-6 spp.) of angiosperms overall, but eelgrass productivity is low (<2 g dw m-2 d-1) and meadows are isolated and genetically impoverished. Higher salinity areas support monospecific meadows, with higher productivity (3-10 g dw m-2 d-1) and greater genetic connectivity. The salinity gradient further imposes functional differences in biodiversity and food webs, in particular a decline in number, but increase in biomass of mesograzers in the Baltic.Significant declines in eelgrass depth limits and areal cover are documented, particularly in regions experiencing high human pressure. The failure of eelgrass to re-establish itself in affected areas, despite nutrient reductions and improved water quality, signals complex recovery trajectories and calls for much greater conservation effort to protect existing meadows.The knowledge base for Nordic eelgrass meadows is broad and sufficient to establish monitoring objectives across nine national borders. Nevertheless, ensuring awareness of their vulnerability remains challenging. Given the areal extent of Nordic eelgrass systems and the ecosystem services they provide, it is crucial to further develop incentives for protecting them. © 2014 The Authors. Aquatic Conservation: Marine and Freshwater Ecosystems published by John Wiley & Sons, Ltd.

Immune suppression of the echinoderm Asterias rubens (L.) following long-term ocean acidification.

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.