Sea anemone-anemonefish symbiosis: Behavior and mucous protein profiling.

Fish species of the genus Amphiprion (Perciformes: Pomacentridae) seek protection from predators among the tentacles of sea anemones as their natural habitat, where they live essentially unharmed from stinging by the host's nematocysts. The skin mucus of these anemonefish has been suggested as a protective mechanism that prevents the discharge of the nematocysts upon contact. Whereas some anemonefish species seem to produce their own protective mucous coating, others may acquire mucus (or biomolecules within) from the sea anemone during an acclimation period. In controlled experiments, we show that Amphiprion ocellaris acclimated successfully to their natural host anemone species Stichodactyla gigantea, and also to Stichodactyla haddoni, and in some cases Heteractis crispa, neither of which are natural host species. No symbiosis was observed for three other anemone species tested, Entacmaea quadricolor, Macrodactyla doreensis, and Heteractis malu. We explored the skin mucous protein profile from naive and experienced A. ocellaris during their acclimation to natural and unnatural host anemones. We confidently report the presence of metabolic and structural proteins in the skin mucus of all samples, likely involved in immunological defense, molecular transport, stress response, and signal transduction. For those anemonefish that established symbiosis, there was a clear increase in ribosomal-type proteins. We additionally provide evidence for the presence of anemone proteins only in the skin mucus of individuals that established symbiosis. Our results support previous speculation of the role of skin mucous-associated proteins in anemonefish-anemone symbiosis. Further exploration of these mucosal proteins could reveal the mechanism of anemonefish acclimation to host anemones.

Growth portfolios buffer climate-linked environmental change in marine systems.

Large-scale, climate-induced synchrony in the productivity of fish populations is becoming more pronounced in the world's oceans. As synchrony increases, a population's "portfolio" of responses can be diminished, in turn reducing its resilience to strong perturbation. Here we argue that the costs and benefits of trait synchronization, such as the expression of growth rate, are context dependent. Contrary to prevailing views, synchrony among individuals could actually be beneficial for populations if growth synchrony increases during favorable conditions, and then declines under poor conditions when a broader portfolio of responses could be useful. Importantly, growth synchrony among individuals within populations has seldom been measured, despite well-documented evidence of synchrony across populations. Here, we used century-scale time series of annual otolith growth to test for changes in growth synchronization among individuals within multiple populations of a marine keystone species (Atlantic cod, Gadus morhua). On the basis of 74,662 annual growth increments recorded in 13,749 otoliths, we detected a rising conformity in long-term growth rates within five northeast Atlantic cod populations in response to both favorable growth conditions and a large-scale, multidecadal mode of climate variability similar to the East Atlantic Pattern. The within-population synchrony was distinct from the across-population synchrony commonly reported for large-scale environmental drivers. Climate-linked, among-individual growth synchrony was also identified in other Northeast Atlantic pelagic, deep-sea and bivalve species. We hypothesize that growth synchrony in good years and growth asynchrony in poorer years reflects adaptive trait optimization and bet hedging, respectively, that could confer an unexpected, but pervasive and stabilizing, impact on marine population productivity in response to large-scale environmental change.

Otolith "spawning zones" across multiple Atlantic cod populations: Do they accurately record maturity and spawning?

Specific changes identified in the otolith macrostructure of Northeast Arctic cod as "spawning zones" are presumed to represent spawning events, but recent experimental studies have challenged this relationship. Because these zones are not routinely recorded outside of Norway, otoliths from multiple Atlantic cod populations with different life history and environmental traits were first examined to see if spawning zones could be identified as a general characteristic of cod. Then, a large archival collection of cod otoliths was used to investigate temporal changes in the occurrence of spawning zones and test for correlations between maturity at age derived from otolith spawning zones and gonad maturity stages. This study shows that spawning zones likely are a universal trait of Atlantic cod and not limited to certain environments or migratory behaviors as previously proposed. Maturity at age derived from spawning zone data showed trends consistent with those from gonad examinations. However, spawning zones appear to form with a one- or two-year lag with sexual maturity, which is suspected to reflect a stabilizing of energy partitioning after the first spawning events. Our results illustrate the potential for use of spawning zones, for example in species or populations with limited available maturity data, and highlights the need for addressing the physiological processes behind their formation.

Differences in metabolic rate between two Atlantic cod (Gadus morhua) populations estimated with carbon isotopic composition in otoliths.

The isotopic composition of inorganic carbon in otoliths (δ13Coto) can be a useful tracer of metabolic rates and a method to study ecophysiology in wild fish. We evaluated environmental and physiological sources of δ13Coto variation in Icelandic and Northeast Arctic (NEA) cod (Gadus morhua) over the years 1914-2013. Individual annual growth increments of otoliths formed at age 3 and 8 were micromilled and measured by isotope-ratio mass spectrometry. Simultaneously, all annual increment widths of the otoliths were measured providing a proxy of fish somatic growth. We hypothesized that changes in the physiological state of the organism, reflected by the isotopic composition of otoliths, can affect the growth rate. Using univariate and multivariate mixed-effects models we estimated conditional correlations between carbon isotopic composition and growth of fish at different levels (within individuals, between individuals, and between years), controlling for intrinsic and extrinsic effects on both otolith measurements. δ13Coto was correlated with growth within individuals and between years, which was attributed to the intrinsic effects (fish age or total length). There was no significant correlation between δ13Coto and growth between individuals, which suggests that caution is needed when interpreting δ13Coto signals. We found a significant decrease in δ13Coto through the century which was explained by the oceanic Suess effect-admixture of isotopically light carbon from fossil fuel. We calculated the proportion of the respired carbon in otolith carbonate (Cresp) using carbon isotopic composition in diet and dissolved inorganic carbon of the seawater. This approach allowed us to correct the values for each stock in relation to these two environmental baselines. Cresp was on average 0.275 and 0.295 in Icelandic and NEA stock, respectively. Our results provide an insight into the physiological basis for differences in growth characteristics between these two cod stocks, and how that may vary over time.

Lemon sole Microstomus kitt in the northern North Sea: a multidisciplinary approach to the early life-history dynamics.

Lemon sole Microstomus kitt is a commercially valuable flatfish species that occurs in shelf waters around the northeast Atlantic. Only the most basic life-history information is available for the North Sea. Spawning is generally assumed to occur between early May and October, with a peak between May and August. Lemon sole larvae have been found in the water column in the northern North Sea in winter during standard surveys. Larvae captured in November/December 2016 and January/February 2017 using the International Council for the Exploration of the Seas standard 2 m Midwater Ring trawls (MIK) were analysed to gain a better understanding of the pelagic early life-history stages of lemon sole, especially in relation to the timing of spawning and the dispersal of overwintering larvae. Larval age was estimated from sagittal otolith primary increment counts. The larvae caught in November/December ranged in nominal age from 4 to 45 days post-hatching which suggests that spawning continues into late October and November. Most, but not all, of the larvae caught in January/February were post metamorphosis, and the difference in age between the two sampling dates was consistent with the elapsed time between samplings. The estimated hatching dates confirm that lemon sole spawning extends into late autumn in the northern North Sea, with overwintering larvae in all developmental stages. Drift modelling of eggs and larvae released at historically documented spawning grounds in the northern North Sea suggests that these grounds are also the source for all of the larvae sampled during the 2016-2017 surveys.

Century-long cod otolith biochronology reveals individual growth plasticity in response to temperature.

Otolith biochronologies combine growth records from individual fish to produce long-term growth sequences, which can help to disentangle individual from population-level responses to environmental variability. This study assessed individual thermal plasticity of Atlantic cod (Gadus morhua) growth in Icelandic waters based on measurements of otolith increments. We applied linear mixed-effects models and developed a century-long growth biochronology (1908-2014). We demonstrated interannual and cohort-specific changes in the growth of Icelandic cod over the last century which were mainly driven by temperature variation. Temperature had contrasting relationships with growth-positive for the fish during the youngest ages and negative during the oldest ages. We decomposed the effects of temperature on growth observed at the population level into within-individual effects and among-individual effects and detected significant individual variation in the thermal plasticity of growth. Variance in the individual plasticity differed across cohorts and may be related to the mean environmental conditions experienced by the group. Our results underscore the complexity of the relationships between climatic conditions and the growth of fish at both the population and individual level, and highlight the need to distinguish between average population responses and growth plasticity of the individuals for accurate growth predictions.

A century of fish growth in relation to climate change, population dynamics and exploitation.

Marine ecosystems, particularly in high-latitude regions such as the Arctic, have been significantly affected by human activities and contributions to climate change. Evaluating how fish populations responded to past changes in their environment is helpful for evaluating their future patterns, but is often hindered by the lack of long-term biological data available. Using otolith increments of Northeast Arctic cod (Gadus morhua) as a proxy for individual growth, we developed a century-scale biochronology (1924-2014) based on the measurements of 3,894 fish, which revealed significant variations in cod growth over the last 91 years. We combined mixed-effect modeling and path analysis to relate these growth variations to selected climate, population and fishing-related factors. Cod growth was negatively related to cod population size and positively related to capelin population size, one of the most important prey items. This suggests that density-dependent effects are the main source of growth variability due to competition for resources and cannibalism. Growth was also positively correlated with warming sea temperatures but negatively correlated with the Atlantic Multidecadal Oscillation, suggesting contrasting effects of climate warming at different spatial scales. Fishing pressure had a significant but weak negative direct impact on growth. Additionally, path analysis revealed that the selected growth factors were interrelated. Capelin biomass was positively related to sea temperature and negatively influenced by herring biomass, while cod biomass was mainly driven by fishing mortality. Together, these results give a better understanding of how multiple interacting factors have shaped cod growth throughout a century, both directly and indirectly.

Host choice and fitness of anemonefish Amphiprion ocellaris (Perciformes: Pomacentridae) living with host anemones (Anthozoa: Actiniaria) in captive conditions.

In this study, we investigated the host choice of naïve Amphiprion ocellaris, a specialist, at two different stages of development (newly settling juveniles and post-settlement juveniles). The fish were exposed to their natural and unnatural host species in the laboratory and their fitness was assessed in terms of activity and growth rate. Newly settling juveniles exhibited little host preference, while post-settlement juveniles immediately associated with their most common host in the wild. The analysis of fish activity confirmed that A. ocellaris is diurnal; they are most active in the morning, less at midday and barely move at night. The average travelling distance of juveniles was shorter in the groups living with their natural host, increasing in the groups living with an unnatural host and was highest in groups that did not become associated with any other unnatural host species. Post-settlement juveniles living with the natural host species grew better than those living with unnatural hosts or without anemone contact. These results suggest that the welfare of A. ocellaris in captivity will be optimized by keeping them with their natural anemone host species, although more generalist Amphiprion species may survive in association with other hosts.

Organ damage in Atlantic herring larvae as a result of ocean acidification.

The dissolution of anthropogenically emitted excess carbon dioxide lowers the pH of the world's ocean water. The larvae of mass spawning marine fishes may be particularly vulnerable to such ocean acidification (OA), yet the generality of earlier results is unclear. Here we show the detrimental effects of OA on the development of a commercially important fish species, the Atlantic herring (Clupea harengus). Larvae were reared at three levels of CO2: today (0.0385 kPa), end of next century (0.183 kPa), and a coastal upwelling scenario (0.426 kPa), under near-natural conditions in large outdoor tanks. Exposure to elevated CO2 levels resulted in stunted growth and development, decreased condition, and severe tissue damage in many organs, with the degree of damage increasing with CO2 concentration. This complements earlier studies of OA on Atlantic cod larvae that revealed similar organ damage but at increased growth rates and no effect on condition.

The proteome of Atlantic herring (Clupea harengus L.) larvae is resistant to elevated pCO2.

Elevated anthropogenic pCO2 can delay growth and impair otolith structure and function in the larvae of some fishes. These effects may concurrently alter the larva's proteome expression pattern. To test this hypothesis, Atlantic herring larvae were exposed to ambient (370 µatm) and elevated (1800 µatm) pCO2 for one-month. The proteome structure of the larvae was examined using a 2-DE and mass spectrometry. The length of herring larvae was marginally less in the elevated pCO2 treatment compared to the control. The proteome structure was also different between the control and treatment, but only slightly: the expression of a small number of proteins was altered by a factor of less than 2-fold at elevated pCO2. This comparative proteome analysis suggests that the proteome of herring larvae is resilient to elevated pCO2. These observations suggest that herring larvae can cope with levels of CO2 projected for near future without significant proteome-wide changes.

Outlier SNP markers reveal fine-scale genetic structuring across European hake populations (Merluccius merluccius).

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome-wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large- and fine-scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F(CT) = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F(CT) range 0.275-0.705) and fine-scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.

A genomic island linked to ecotype divergence in Atlantic cod.

The genomic architecture underlying ecological divergence and ecological speciation with gene flow is still largely unknown for most organisms. One central question is whether divergence is genome-wide or localized in 'genomic mosaics' during early stages when gene flow is still pronounced. Empirical work has so far been limited, and the relative impacts of gene flow and natural selection on genomic patterns have not been fully explored. Here, we use ecotypes of Atlantic cod to investigate genomic patterns of diversity and population differentiation in a natural system characterized by high gene flow and large effective population sizes, properties which theoretically could restrict divergence in local genomic regions. We identify a genomic region of strong population differentiation, extending over approximately 20 cM, between pairs of migratory and stationary ecotypes examined at two different localities. Furthermore, the region is characterized by markedly reduced levels of genetic diversity in migratory ecotype samples. The results highlight the genomic region, or 'genomic island', as potentially associated with ecological divergence and suggest the involvement of a selective sweep. Finally, we also confirm earlier findings of localized genomic differentiation in three other linkage groups associated with divergence among eastern Atlantic populations. Thus, although the underlying mechanisms are still unknown, the results suggest that 'genomic mosaics' of differentiation may even be found under high levels of gene flow and that marine fishes may provide insightful model systems for studying and identifying initial targets of selection during ecological divergence.

Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Frequent skipped spawning in the world's largest cod population.

Life-history theory suggests that animals may skip reproductive events after initial maturation to maximize lifetime fitness. In iteroparous teleosts, verifying past spawning history is particularly difficult; the degree of skipped spawning at the population level therefore remains unknown. We unequivocally show frequent skipped spawning in Northeast Arctic cod (NEAC) in a massive field and laboratory effort from 2006 to 2008. This was verified by postovulatory follicles in temporarily arrested ovaries close to the putative spawning period. At the population level, "skippers" were estimated to be approximately equally abundant as spawning females in 2008, constituting ∼24% of the females 60-100 cm. These females never truly started vitellogenesis and principally remained on the feeding grounds when spawners migrated southward, avoiding any migration costs. The proximate cause of skipping seems to be insufficient energy to initiate oocyte development, indicating that skipped spawning may partly be a density-dependent response important in population regulation. Our data also indicate more skipping among smaller females and potential tradeoffs between current and future reproductive effort. We propose that skipped spawning is an integral life-history component for NEAC, likely varying annually, and it could therefore be an underlying factor causing some of the currently unexplained large NEAC recruitment variation. The same may hold for other teleosts.

Low-dose exposure to alkylphenols adversely affects the sexual development of Atlantic cod (Gadus morhua): acceleration of the onset of puberty and delayed seasonal gonad development in mature female cod.

Produced water (PW), a by-product of the oil-production process, contains large amount of alkylphenols (APs) and other harmful oil compounds. In the last 20 years, there have been increasing concerns regarding the environmental impact of large increases in the amounts of PW released into the North Sea. We have previously shown that low levels of APs can induce disruption of the endocrine and reproductive systems of Atlantic cod (Gadus morhua). The aims of this follow-up study were to: (i) identify the lowest observable effect concentration of APs; (ii) study the effects of exposure to real PW, obtained from a North Sea oil-production platform; and (iii) study the biological mechanism of endocrine disruption in female cod. Fish were fed with feed paste containing several concentrations of four different APs (4-tert-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol) or real PW for 20 weeks throughout the normal period of vitellogenesis in Atlantic cod from October to January. Male and female cod, exposed to AP and PW, were compared to unexposed fish and to fish fed paste containing 17ß-oestradiol (E(2)). Approximately 60% of the females and 96% of the males in the unexposed groups were mature at the end of the experiment. Our results show that exposure to APs and E(2) have different effects depending on the developmental stage of the fish. We observed that juvenile females are advanced into puberty and maturation, while gonad development was delayed in both maturing females and males. The AP-exposed groups contained increased numbers of mature females, and significant differences between the untreated group and the AP-treated groups were seen down to a dose of 4 µg AP/kg body weight. In the high-dose AP and the E(2) exposed groups, all females matured and no juveniles were seen. These results suggest that AP-exposure can affect the timing of the onset of puberty in fish even at extremely low concentrations. Importantly, similar effects were not seen in the fish that were exposed to real PW.

Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.