Charakterisierung von Arthropodenhämocyaninen

Verschiedene cDNA-Banken wurden erstellt, andere waren bereits vorhanden. Diese Expressionsbanken wurden mittels Antikörper- bzw. Sondenscreening auf Hämocyanin- bzw. bei den Insekten auch auf Hexamerinklone durchmustert. Dabei gelang es, diverse Hämocyaninunter-einheiten zu identifizieren und zu sequenzieren. Die erarbeiteten Sequenzen wurden im An-schluss für phylogenetische Studien benutzt. Es konnte hierbei gezeigt werden, dass die so-genannte „Tracheata-Hypothese“ immer unwahrscheinlicher wird. Diese geht von einer Schwestergruppenstellung der Hexapoda und der Myriapoda aus. Die hier gemachten Unter-suchungen widersprechen dieser Hypothese eindeutig. Die Ergebnisse stützen vielmehr die „Pancrustacea-Hypothese“. In den Stammbäumen sind die Crustaceenhämocyanine eindeutig in naher Verwandtschaft zu den paraphyletisch entstandenen Insektenhämocyaninen angeordnet. Ob die Crustaceen monophyletischen Ursprungs sind, kann nicht eindeutig beurteilt werden, es bestehen aber weiterhin begründete Zweifel. Die Myriapodenhämocyanine stehen in Schwestergruppenstellung zu den Crustaceen und den Insektenhämocyaninen. Dies weist auf ein gemeinsames Taxon der Mandibulata hin. Die systematische Stellung der Myriapoden innerhalb der Arthropoden kann allerdings nicht abschließend geklärt werden. Dazu ist eine weitergehende Aufklärung von Hämocyaninsequenzen in diesem Arthropodentaxon notwendig.

10 Å cryo-EM structure of Drosophila melanogaster hexamerin and Limulus polyphemus hemocyanin

In the present study, the quaternary structures of Drosophila melanogaster hexamerin LSP-2 and Limulus polyphemus hemocyanin, both proteins from the hemocyanin superfamily, were elucidated to a 10 Å resolution with the technique of cryo-EM 3D-reconstruction. Furthermore, molecular modelling and rigid-body fitting allowed a detailed insight into the cryo-EM structures at atomic level. The results are summarised as follows: Hexamerin 1. The cryo-EM structure of Drosophila melanogaster hexamerin LSP-2 is the first quaternary structure of a protein from the group of the insect storage proteins. 2. The hexamerin LSP-2 is a hexamer of six bean-shaped subunits that occupy the corners of a trigonal antiprism, yielding a D3 (32) point-group symmetry. 3. Molecular modelling and rigid-body fitting of the hexamerin LSP-2 sequence showed a significant correlation between amino acid inserts in the primary structure and additional masses of the cryo-EM structure that are not present in the published quaternary structures of chelicerate and crustacean hemocyanins. 4. The cryo-EM structure of Drosophila melanogaster hexamerin LSP-2 confirms that the arthropod hexameric structure is applicable to insect storage proteins. Hemocyanin 1. The cryo-EM structure of the 8×6mer Limulus polyphemus hemocyanin is the highest resolved quaternary structure of an oligo-hexameric arthropod hemocyanin so far. 2. The hemocyanin is build of 48 bean-shaped subunits which are arranged in eight hexamers, yielding an 8×6mer with a D2 (222) point-group symmetry. The 'basic building blocks' are four 2×6mers that form two 4×6mers in an anti-parallel manner, latter aggregate 'face-to-face' to the 8×6mer. 3. The morphology of the 8×6mer was gauged and described very precisely on the basis of the cryo-EM structure. 4. Based on earlier topology studies of the eight different subunit types of Limulus polyphemus hemocyanin, eleven types of interhexamer interfaces have been identified that in the native 8×6mer sum up to 46 inter-hexamer bridges - 24 within the four 2×6mers, 10 to establish the two 4×6mers, and 12 to assemble the two 4×6mers into an 8×6mer. 5. Molecular modelling and rigid-body fitting of Limulus polyphemus and orthologous Erypelma californicum sequences allowed to assign very few amino acids to each of these interfaces. These amino acids now serve as candidates for the chemical bonds between the eight hexamers. 6. Most of the inter-hexamer contacts are conspicuously histidine-rich and evince constellations of amino acids that could constitute the basis for the allosteric interactions between the hexamers. 7. The cryo-EM structure of Limulus polyphemus hemocyanin opens the door to a fundamental understanding of the function of this highly cooperative protein.

Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation.

Megistobenthic faunal diversity of the Antalya Gulf: Crustacea

The spatio-temporal distribution of megistobenthic crustacean assemblages from the Antalya Gulf, located in the Levantine Sea is described. In order to provide a comprehensive overview of the spatio-temporal patterns of the crustacean community, 3 transect including depth of 10, 25, 75, 125 and 200 m, were studied between 2014 and 2015 to investigate their association with a set of environmental parameters in representative months of each season (spring, summer, autumn and winter). For its economic importance in Levantine waters, a focus analysis of deep-water rose shrimp Parapenaeus longirostris (Lucas, 1846) was done, to investigate the length frequency composition of the population of the Antalya Gulf. A total of 58 crustacean species were encountered in the study area, of these species identified, 18 species were recognized as alien species in the Mediterranean Sea. Throughout the year the most frequent species of the study were the hermit crab Pagurus prideaux (Leach, 1815) and Parapenaeus longirostris (Lucas, 1846) followed by the Indo-Pacific swimming crab Charybdis longicollis (Leene, 1938) and by the invasive shrimp Marsupenaeus japonicus (Spence Bate, 1888). Few species contributing to a high amount to the total biomass were found throughout the year. These species were Charybdis longicollis and Parapenaeus longirostris. Stations of the study area showed similar values of diversity indices of benthic crustacean community among the three transect. The highest values of faunistic indices were detected in autumn and winter (October and February), and also varied along the depth gradient, with the highest values found between 25 and 75 meters. The multivariate analyses conducted on the abundance data point out major differences between depths and between seasons. Therefore, according to cluster analysis and ordination over abundance and biomass, three main crustacean assemblages were detected: the first corresponding to shallow bottoms (10, 25 meters), the second corresponding to intermediate waters (75 meters) and the last to deeper waters (125, 200 meters). Depth was the main factor governing the distribution of megistobenthic crustacean in the area. Besides the depth, the structure of the sediment is the most important factor in determining the crustacean assemblage. Therefore, all factors governing the crustacean distribution were found to be related to the bottom depth. The population of Parapenaeus longirostris in the Antalya Gulf showed significant differences in depth. It was found that females dominated the population of the study area (65.11%), and were significantly larger than males for each cohort identified. The size-weight relationships revealed a slight negative allometry in growth, a bit more pronounced in females than in males.

Factors Affecting Post-Capture Survivability of Lobster Homarus Americanus

Technological advances in gear and fishing practices have driven the global expansion of the American lobster live seafood market. These changes have had a positive effect on the lobster industry by increasing capture efficiency. However, it is unknown what effect these improved methods will have on the post-capture fitness and survival of lobsters. This project utilized a repeated measures design to compare the physiological changes that occur in lobsters over time as the result of differences in depth, hauling rate, and storage methodology. The results indicate that lobsters destined for long distance transport or temporary storage in pounds undergo physiological disturbance as part of the capture process. These changes are significant over time for total hemocyte counts, crustacean hyperglycemic hormone, L-lactate, ammonia, and glucose. Repeated measures multivariate analysis of variance (MANOVA) for glucose indicates a significant interaction between depth and storage methodology over time for non-survivors. A Gram-negative bacterium, Photobacterium indicum, was identified in pure culture from hemolymph samples of 100% of weak lobsters. Histopathology revealed the presence of Gram-negative bacteria throughout the tissues with evidence of antemortem edema and necrosis suggestive of septicemia. On the basis of these findings, we recommend to the lobster industry that if a reduction in depth and hauling rate is not economically feasible, fishermen should take particular care in handling lobsters and provide them with a recovery period in recirculating seawater prior to land transport. The ecological role of P. indicum is not fully defined at this time. However, it may be an emerging opportunistic pathogen of stressed lobsters. Judicious preemptive antibiotic therapy may be necessary to reduce mortality in susceptible lobsters destined for high-density holding facilities.

Structures and Concentrations of Surfactants in Gut Fluid of the Marine Polychaete Arenicola Marina

Marine invertebrate deposit feeders secrete surfactants into their gut fluid in concentrations sufficient to induce micelle formation, enhancing solubilization of sedimentary lipids. We isolated and identified 3 related surfactant molecules from the deposit-feeding polychaete lugworm Arenicola marina. Surfactants were isolated and separated by a combination of solvent extraction and thin-layer and gas chromatography. Identification was performed using mass and infrared spectrometry, coupled to various derivatization and hydrolysis reactions. A. marina produces a mixture of related yet distinct anionic surfactants composed of branched, C9, saturated and unsaturated fatty acids that are amide linked to leucine or glycine residues, showing some similarity to crustacean surfactants. The critical micelle concentration of the mixture of these surfactants in gut fluid was about 2 mM, and total concentrations ranged from 5.5 to 19.5 mM. The hydrophilic amide linkage helps to explain previous observations that gut surfactants do not adsorb onto sediment transiting the gut.

The activation peptide of coagulation factor XIII is vital for its expression and stability.

BACKGROUND The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

Abundance and biomass of dinoflagellates and ciliates at time series station Helgoland Roads, North Sea, 2007-2009

A monitoring programme for microzooplankton was started at the long-term sampling station ''Kabeltonne'' at Helgoland Roads (54°11.30' N; 7°54.00' E) in January 2007 in order to provide more detailed knowledge on microzooplankton occurrence, composition and seasonality patterns at this site and to complement the existing plankton data series. Ciliate and dinoflagellate cell concentration and carbon biomass were recorded on a weekly basis. Heterotrophic dinoflagellates were considerably more important in terms of biomass than ciliates, especially during the summer months. However, in early spring, ciliates were the major group of microzooplankton grazers as they responded more quickly to phytoplankton food availability. Mixotrophic dinoflagellates played a secondary role in terms of biomass when compared to heterotrophic species; nevertheless, they made up an intense late summer bloom in 2007. The photosynthetic ciliate Myrionecta rubra bloomed at the end of the sampling period. Due to its high biomass when compared to crustacean plankton especially during the spring bloom, microzooplankton should be regarded as the more important phytoplankton grazer group at Helgoland Roads. Based on these results, analyses of biotic and abiotic factors driving microzooplankton composition and abundance are necessary for a full understanding of this important component of the plankton.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and magnesium in adult Paralomis granulosa (Decapoda, Anomura, Lithodidae)

A low capacity for regulation of extracellular Mg2+ has been proposed to exclude reptant marine decapod crustaceans from temperatures below 0°C and thus to exclude them from the high Antarctic. To test this hypothesis and to elaborate the underlying mechanisms in the most cold-tolerant reptant decapod family of the sub-Antarctic, the Lithodidae, thermal tolerance was determined in the crab Paralomis granulosa (Decapoda, Anomura, Lithodidae) using an acute stepwise temperature protocol (-1°, 1°, 4°, 7°, 10°, and 13°C). Arterial and venous oxygen partial pressures (Po2) in hemolymph, heartbeat and ventilation beat frequencies, and hemolymph cation composition were measured at rest and after a forced activity (righting) trial. Scopes for heartbeat and ventilation beat frequencies and intermittent heartbeat and scaphognathite beat rates at rest were evaluated. Hemolymph [Mg2+] was experimentally reduced from 30 mmol/L to a level naturally observed in Antarctic caridean shrimps (12 mmol/L) to investigate whether the animals remain more active and tolerant to cold (-1°, 1°, and 4°C). In natural seawater, righting speed was significantly slower at -1° and 13°C, compared with acclimation temperature (4°C). Arterial and venous hemolymph Po2 increased in response to cooling even though heartbeat and ventilation beat frequencies as well as scopes decreased. At rest, ionic composition of the hemolymph was not affected by temperature. Activity induced a significant increase in hemolymph [K+] at -1° and 1°C. Reduction of hemolymph [Mg2+] did not result in an increase in activity, an increase in heartbeat and ventilation beat frequencies, or a shift in thermal tolerance to lower temperatures. In conclusion, oxygen delivery in this cold-water crustacean was not acutely limiting cold tolerance, and animals may have been constrained more by their functional capacity and motility. In contrast to earlier findings in temperate and subpolar brachyuran crabs, these constraints remained insensitive to changing Mg2+ levels.

Zooplankton abundance for two shelf stations off Galicia (NW Spain) from 1994 to 2006

Zooplankton was sampled by project RADIALES at Vigo (E3VI) and A Coruña (E2CO) between 1994 and 2006. Samples were collected using 50-cm diameter Juday-Bogorov (A Coruña) or 40-cm diameter bongo plankton nets (Vigo) equipped with 200-µm mesh size. Tows were double oblique from surface to near bottom (90 and 70 m in Vigo and A Coruña, respectively). All samples were collected between 10:00 and 14:00 o'clock (local time). Samples were preserved in 2-4% sodium borate-buffered formaldehyde. For the purpose of this study, the original coastal time series were categorized in copepods representative of crustacean zooplankton) and gelatinous plankton (medusae and tunicates). Medusae included Hydrozoans and Scyphozoa, and tunicates included salps, pyrosomes, doliolids, and appendicularia. Plankton identification and counts were performed by Ana Miranda and M. Teresa Álvarez-Ossorio for samples from Vigo and A Coruña, respectively. Different trends were found for gelatinous plankton in the two coastal sites, characterized by increases in either medusae or tunicates. Multiyear periods of relative dominance of gelatinous vs. copepod plankton were evident. In general, copepod periods were observed in positive phases of the main modes of regional climatic variability. Conversely, gelatinous periods occurred during negative climatic phases. However, the low correlations between gelatinous plankton and either climatic, oceanographic, or fishery variables suggest that local factors play a major role in their proliferations.

Literature review of elasmobranch bycatch and retention rate

To address growing concern over the effects of fisheries non-target catch on elasmobranchs worldwide, the accurate reporting of elasmobranch catch is essential. This requires data on a combination of measures, including reported landings, retained and discarded non-target catch, and post-discard survival. Identification of the factors influencing discard vs. retention is needed to improve catch estimates and to determine wasteful fishing practices. To do this we compared retention rates of elasmobranch non-target catch in a broad subset of fisheries throughout the world by taxon, fishing country, and gear. A regression tree and random forest analysis indicated that taxon was the most important determinant of retention in this dataset, but all three factors together explained 59% of the variance. Estimates of total elasmobranch removals were calculated by dividing the FAO global elasmobranch landings by average retention rates and suggest that total elasmobranch removals may exceed FAO reported landings by as much as 400%. This analysis is the first effort to directly characterize global drivers of discards for elasmobranch non-target catch. Our results highlight the importance of accurate quantification of retention and discard rates to improve assessments of the potential impacts of fisheries on these species.

Results from experiments and investigations on the kelp crab Taliepus dentatus

Studies of thermal tolerance in marine ectotherms are key in understanding climate effects on ecosystems; however, tolerance of their larval stages has rarely been analyzed. Larval stages are expected to be particularly sensitive. Thermal stress may affect their potential for dispersal and zoogeographical distribution. A mismatch between oxygen demand and the limited capacity of oxygen supply to tissues has been hypothesized to be the first mechanism restricting survival at thermal extremes. Therefore, thermal tolerance of stage zoea I larvae was examined in two populations of the Chilean kelp crab Taliepus dentatus, which are separated by latitude and the thermal regime. We measured temperature-dependent activity, oxygen consumption, cardiac performance, body mass and the carbon (C) and nitrogen (N) composition in order to: (1) examine thermal effects from organismal to cellular levels, and (2) compare the thermal tolerance of larvae from two environmental temperature regimes. We found that larval performance is affected at thermal extremes indicated by decreases in activity, mainly in maxilliped beat rates, followed by decreases in oxygen consumption rates. Cardiac stroke volume was almost temperature-independent. Through changes in heart rate, cardiac output supported oxygen demand within the thermal window whereas at low and high temperature extremes heart rate declined. The comparison between southern and central populations suggests the adaptation of southern larvae to a colder temperature regime, with higher cardiac outputs due to increased cardiac stroke volumes, larger body sizes but similar body composition as indicated by similar C:N ratios. This limited but clear differentiation of thermal windows between populations allows the species to widen its biogeographical range.