Proteomic fingerprinting enables quantitative biodiversity assessments of species and ontogenetic stages in Calanus congeners (Copepoda, Crustacea) from the Arctic Ocean.

Species identification is pivotal in biodiversity assessments and proteomic fingerprinting by MALDI-TOF mass spectrometry has already been shown to reliably identify calanoid copepods to species level. However, MALDI-TOF data may contain more information beyond mere species identification. In this study, we investigated different ontogenetic stages (copepodids C1-C6 females) of three co-occurring Calanus species from the Arctic Fram Strait, which cannot be identified to species level based on morphological characters alone. Differentiation of the three species based on mass spectrometry data was without any error. In addition, a clear stage-specific signal was detected in all species, supported by clustering approaches as well as machine learning using Random Forest. More complex mass spectra in later ontogenetic stages as well as relative intensities of certain mass peaks were found as the main drivers of stage distinction in these species. Through a dilution series, we were able to show that this did not result from the higher amount of biomass that was used in tissue processing of the larger stages. Finally, the data were tested in a simulation for application in a real biodiversity assessment by using Random Forest for stage classification of specimens absent from the training data. This resulted in a successful stage-identification rate of almost 90%, making proteomic fingerprinting a promising tool to investigate polewards shifts of Atlantic Calanus species and, in general, to assess stage compositions in biodiversity assessments of Calanoida, which can be notoriously difficult using conventional identification methods.

Dietary preferences of brachyuran crabs from Taiwan for marine or terrestrial food sources: evidence based on fatty acid trophic markers.

BACKGROUND: Trophic interactions are key processes, which determine the ecological function and performance of organisms. Many decapod crustaceans feed on plant material as a source for essential nutrients, e.g. polyunsaturated fatty acids. Strictly herbivorous feeding appears only occasionally in marine decapods but is common in land crabs. To verify food preferences and to establish trophic markers, we studied the lipid and fatty acid composition of the midgut glands of two marine crab species (Grapsus albolineatus and Percnon affine), one semi-terrestrial species (Orisarma intermedium, formerly Sesarmops intermedius), and one terrestrial species (Geothelphusa albogilva) from Taiwan. RESULTS: All species showed a wide span of total lipid levels ranging from 4 to 42% of the dry mass (%DM) in the marine P. affine and from 3 to 25%DM in the terrestrial G. albogilva. Triacylglycerols (TAG) were the major storage lipid compound. The fatty acids 16:0, 18:1(n-9), and 20:4(n-6) prevailed in all species. Essential fatty acids such as 20:4(n-6) originated from the diet. Terrestrial species also showed relatively high amounts of 18:2(n-6), which is a trophic marker for vascular plants. The fatty acid compositions of the four species allow to clearly distinguish between marine and terrestrial herbivorous feeding due to significantly different amounts of 16:0, 18:1(n-9), and 18:2(n-6). CONCLUSIONS: Based on the fatty acid composition, marine/terrestrial herbivory indices were defined and compared with regard to their resolution and differentiating capacity. These indices can help to reveal trophic preferences of unexplored species, particularly in habitats of border regions like mangrove intertidal flats and estuaries.

Shell disease does not affect biochemical profiles of the North Sea brown shrimp Crangon crangon.

The brown shrimp Crangon crangon is a key component of the North Atlantic coastal food web and an important target species for the fishery economy. As the brown shrimp contains large amounts of protein and essential fatty acids, its consumption makes it a beneficial choice for humans. Commercially harvested crustaceans like C. crangon are frequently affected by bacterial shell disease, with necrotizing erosions and ulcerations of the cuticle. To determine whether shell disease influences the nutritional value of C. crangon, total protein and lipid contents, as well as fatty acid compositions of muscle tissue and hepatopancreas, together with the hepatosomatic index, were examined in healthy and affected individuals. The biochemical composition of the tissues did not differ significantly between the 2 groups. Also, the hepatosomatic index, as an indicator of energy reserves in shrimps, was similar between healthy and affected animals. Our results indicate that the nutritional value of C. crangon is not affected by shell disease, as long as it remains superficial as in the present study.

Molecular aspects of lipid metabolism in the midgut gland of the brown shrimp Crangon crangon.

The brown shrimp, Crangon crangon, is well adapted to the variable environmental conditions in the southern North Sea. It is very abundant, has high reproduction rates, and holds a key position in coastal ecosystems. This species has very low lipid deposits in the midgut gland, suggesting that the main function of the midgut gland is metabolic turnover rather than energy storage. Based on seasonal gene expression studies and established transcriptome data, we investigated key components of lipid metabolic pathways. Gene expression of triacylglycerol lipase, phospholipase, and fatty acid desaturase were analyzed and compared with that of other digestive enzymes involved in lipid, carbohydrate, and protein catabolism. Our results suggest that gene expression of digestive enzymes involved in lipid metabolism is modulated by the lipid content in the midgut gland and is related to food availability. Brown shrimp seem to be capable of using cellular phospholipids during periods of food paucity but high energetic (lipid) requirements. Two of three isoforms of fatty acid binding proteins (FABPs) from the midgut gland involved in fatty acid transport showed specific mutations of the binding site. We hypothesize that the mutations in FABPs and deficiencies in anabolic pathways limit lipid storage capacities in the midgut gland of C. crangon. In turn, food utilization, including lipid catabolism, has to be efficient to fulfill the energetic requirements of brown shrimp.

Assimilation and turnover rates of lipid compounds in dominant Antarctic copepods fed with 13C-enriched diatoms.

The study revealed species- and stage-specific differences in lipid accumulation of the dominant Antarctic copepods, the primarily herbivorous Calanoides acutus (copepodite stage V (CV), females) and the more omnivorous Calanus propinquus (females) storing wax esters and triacylglycerols, respectively, which were collected in summer (end of December). Feeding carbon-labelled diatoms to these copepods, 13C elucidated assimilation and turnover rates of copepod total lipids as well as specific fatty acids and alcohols. The 13C incorporation was monitored by compound-specific stable isotope analysis (CSIA). CV stages of C. acutus exhibited an intense total lipid turnover and 55% of total lipids were labelled after 9 days of feeding. By contrast, total lipid assimilation of female C. acutus and C. propinquus was lower with 29% and 32%, respectively. The major dietary fatty acids 16:0, 16:1(n - 7) and 20:5(n - 3) had high turnover rates in all specimens. In C. acutus CV, the high rates of the de novo synthesized long-chain monounsaturated fatty acids and alcohols 20:1(n - 9) and 22:1(n - 11) indicate intense lipid deposition, whereas these rates were low in females. The differences in lipid assimilation and turnover clearly show that the copepod species exhibit a high variability and plasticity to adapt their lipid production to their various life phases. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Predatory zooplankton on the move: Themisto amphipods in high-latitude marine pelagic food webs.

Hyperiid amphipods are predatory pelagic crustaceans that are particularly prevalent in high-latitude oceans. Many species are likely to have co-evolved with soft-bodied zooplankton groups such as salps and medusae, using them as substrate, for food, shelter or reproduction. Compared to other pelagic groups, such as fish, euphausiids and soft-bodied zooplankton, hyperiid amphipods are poorly studied especially in terms of their distribution and ecology. Hyperiids of the genus Themisto, comprising seven distinct species, are key players in temperate and cold-water pelagic ecosystems where they reach enormous levels of biomass. In these areas, they are important components of marine food webs, and they are major prey for many commercially important fish and squid stocks. In northern parts of the Southern Ocean, Themisto are so prevalent that they are considered to take on the role that Antarctic krill play further south. Nevertheless, although they are around the same size as krill, and may also occur in swarms, their feeding behaviour and mode of reproduction are completely different, hence their respective impacts on ecosystem structure differ. Themisto are major predators of meso- and macrozooplankton in several major oceanic regions covering shelves to open ocean from the polar regions to the subtropics. Based on a combination of published and unpublished occurrence data, we plot out the distributions of the seven species of Themisto. Further, we consider the different predators that rely on Themisto for a large fraction of their diet, demonstrating their major importance for higher trophic levels such as fish, seabirds and mammals. For instance, T. gaudichaudii in the Southern Ocean comprises a major part of the diets of around 80 different species of squid, fish, seabirds and marine mammals, while T. libellula in the Bering Sea and Greenland waters is a main prey item for commercially exploited fish species. We also consider the ongoing and predicted range expansions of Themisto species in light of environmental changes. In northern high latitudes, sub-Arctic Themisto species are replacing truly Arctic, ice-bound, species. In the Southern Ocean, a range expansion of T. gaudichaudii is expected as water masses warm, impacting higher trophic levels and biogeochemical cycles. We identify the many knowlegde gaps that must be filled in order to evaluate, monitor and predict the ecological shifts that will result from the changing patterns of distribution and abundance of this important pelagic group.

Transcriptome analysis of the midgut gland of the brown shrimp Crangon crangon indicates high polymorphism in digestive enzymes.

Tolerance of organisms towards heterogeneous and variable environments is highly related to physiological flexibility. An effective strategy to enhance physiological flexibility is the expression of polymorphic enzymes. This seems to be the case in the brown shrimp Crangon crangon. It shows high reproduction rates, feeds opportunistically on endo- and epibenthic organisms, and is apparently well adapted to variable environmental conditions. Previous electrophoretic studies revealed a high level of polymorphism and no consistent phenotype of digestive enzymes between individuals. In order to understand the underlying biochemical processes, we carried out a transcriptome-based study of digestive enzymes of C. crangon. Detailed sequence analyses of triacylglycerol lipase, phospholipase A2, alpha amylase, chitinase, trypsin and cathepsin L were performed to identify putative isoforms. The number of isoforms, and thus the degree of polymorphism varied among enzymes: lipases and carbohydrases showed higher numbers of isoforms in enzymes that besides their extracellular function also have diverse intracellular functions. Furthermore, cysteine proteinases showed a lower polymorphism than serine proteinases. We suggest that the expression of enzyme isoforms improves the efficiency of C. crangon in gaining energy from different food sources.

Short- and long-term acclimation patterns of the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) along a depth gradient.

The giant kelp, Macrocystis pyrifera, is exposed to highly variable irradiance and temperature regimes across its geographic and vertical depth gradients. The objective of this study was to extend our understanding of algal acclimation strategies on different temporal scales to those varying abiotic conditions at various water depths. Different acclimation strategies to various water depths (0.2 and 4 m) between different sampling times (Jan/Feb and Aug/Sept 2012; long-term acclimation) and more rapid adjustments to different depths (0.2, 2 and 4 m; short-term acclimation) during 14 d of transplantation were found. Adjustments of variable Chl a fluorescence, pigment composition (Chl c, fucoxanthin), and the de-epoxidation state of the xanthophyll cycle pigments were responsible for the development of different physiological states with respect to various solar radiation and temperature climates. Interestingly, the results indicated that phlorotannins are important during long-term acclimation while antioxidants have a crucial role during short-term acclimation. Furthermore, the results suggested that modifications in total lipids and fatty acid compositions apparently also might play a role in depth acclimation. In Aug/Sept (austral winter), M. pyrifera responded to the transplantation from 4 m to 0.2 m depth with a rise in the degree of saturation and a switch from shorter- to longer-chain fatty acids. These changes seem to be essential for the readjustment of thylakoid membranes and might, thus, facilitate efficient photosynthesis under changing irradiances and temperatures. Further experiments are needed to disentangle the relative contribution of solar radiation, temperature and also other abiotic parameters in the observed physiological changes.

Spatio-temporal variability of copepod abundance along the 20 °S monitoring transect in the Northern Benguela upwelling system from 2005 to 2011.

Long-term data sets are essential to understand climate-induced variability in marine ecosystems. This study provides the first comprehensive analysis of longer-term temporal and spatial variations in zooplankton abundance and copepod community structure in the northern Benguela upwelling system from 2005 to 2011. Samples were collected from the upper 200 m along a transect at 20 °S perpendicular to the coast of Namibia to 70 nm offshore. Based on seasonal and interannual trends in surface temperature and salinity, three distinct time periods were discernible with stronger upwelling in spring and extensive warm-water intrusions in late summer, thus, high temperature amplitudes, in the years 2005/06 and 2010/11, and less intensive upwelling followed by weaker warm-water intrusions from 2008/09 to 2009/10. Zooplankton abundance reflected these changes with higher numbers in 2005/06 and 2010/11. In contrast, zooplankton density was lower in 2008/09 and 2009/10, when temperature gradients from spring to late summer were less pronounced. Spatially, copepod abundance tended to be highest between 30 and 60 nautical miles off the coast, coinciding with the shelf break and continental slope. The dominant larger calanoid copepods were Calanoides carinatus, Metridia lucens and Nannocalanus minor. On all three scales studied, i.e. spatially from the coast to offshore waters as well as temporally, both seasonally and interannually, maximum zooplankton abundance was not coupled to the coldest temperature regime, and hence strongest upwelling intensity. Pronounced temperature amplitudes, and therefore strong gradients within a year, were apparently important and resulted in higher zooplankton abundance.

Distribution and ecophysiology of calanoid copepods in relation to the oxygen minimum zone in the eastern tropical atlantic.

Oxygen minimum zones (OMZs) affect distribution patterns, community structure and metabolic processes of marine organisms. Due to the prominent role of zooplankton, especially copepods, in the marine carbon cycle and the predicted intensification and expansion of OMZs, it is essential to understand the effects of hypoxia on zooplankton distribution and ecophysiology. For this study, calanoid copepods were sampled from different depths (0-1800 m) at eight stations in the eastern tropical Atlantic (3 °47'N to 18 °S) during three expeditions in 2010 and 2011. Their horizontal and vertical distribution was determined and related to the extent and intensity of the OMZ, which increased from north to south with minimum O2 concentrations (12.7 µmol kg(-1)) in the southern Angola Gyre. Calanoid copepod abundance was highest in the northeastern Angola Basin and decreased towards equatorial regions as well as with increasing depth. Maximum copepod biodiversity was observed in the deep waters of the central Angola Basin. Respiration rates and enzyme activities were measured to reveal species-specific physiological adaptations. Enzyme activities of the electron transport system (ETS) and lactate dehydrogenase (LDH) served as proxies for aerobic and anaerobic metabolic activity, respectively. Mass-specific respiration rates and ETS activities decreased with depth of occurrence, consistent with vertical changes in copepod body mass and ambient temperature. Copepods of the families Eucalanidae and Metridinidae dominated within the OMZ. Several of these species showed adaptive characteristics such as lower metabolic rates, additional anaerobic activity and diel vertical migration that enable them to successfully inhabit hypoxic zones.

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae).

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81-82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-beta-1,4-glucanase, beta-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-beta-1,4-glucanase and beta-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-beta-D-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

Fatty acid trophic markers in the pelagic marine environment.

Fatty acids have been used as qualitative markers to trace or confirm predator-prey relationships in the marine environment for more than thirty years. More recently, they have also been used to identify key processes impacting the dynamics of some of the world's major ecosystems. The fatty acid trophic marker (FATM) concept is based on the observation that marine primary producers lay down certain fatty acid patterns that may be transferred conservatively to, and hence can be recognized in, primary consumers. To identify these fatty acid patterns the literature was surveyed and a partial least squares (PLS) regression analysis of the data was performed, validating the specificity of particular microalgal FATM. Microalgal group specific FATM have been traced in various primary consumers, particularly in herbivorous calanoid copepods, which accumulate large lipid reserves, and which dominate the zooplankton biomass in high latitude ecosystems. At higher trophic levels these markers of herbivory are obscured as the degree of carnivory increases, and as the fatty acids originate from a variety of dietary sources. Such differences are highlighted in a PLS regression analysis of fatty acid and fatty alcohol compositional data (the components of wax esters accumulated by many marine organisms) of key Arctic and Antarctic herbivorous, omnivorous and carnivorous copepod species. The analysis emphasizes how calanoid copepods separate from other copepods not only by their content of microalgal group specific FATM, but also by their large content of long-chain monounsaturated fatty acids and alcohols. These monounsaturates have been used to trace and resolve food web relationships in, for example, hyperiid amphipods, euphausiids and fish, which may consume large numbers of calanoid copepods. Results like these are extremely valuable for enabling the discrimination of specific prey species utilized by higher trophic level omnivores and carnivores without the employment of invasive techniques, and thereby for identifying the sources of energetic reserves. A conceptual model of the spatial and temporal dominance of group-specific primary producers, and hence the basic fatty acid patterns available to higher trophic levels is presented. The model is based on stratification, which acts on phytoplankton group dominance through the availability of light and nutrients. It predicts the seasonal and ecosystem specific contribution of diatom and flagellate/microbial loop FATM to food webs as a function of water column stability. Future prospects for the application of FATM in resolving dynamic ecosystem processes are assessed.