Deep-sea predator niche segregation revealed by combined cetacean biologging and eDNA analysis of cephalopod prey.

Fundamental insight on predator-prey dynamics in the deep sea is hampered by a lack of combined data on hunting behavior and prey spectra. Deep-sea niche segregation may evolve when predators target specific prey communities, but this hypothesis remains untested. We combined environmental DNA (eDNA) metabarcoding with biologging to assess cephalopod community composition in the deep-sea foraging habitat of two top predator cetaceans. Risso's dolphin and Cuvier's beaked whale selectively targeted distinct epi/meso- and bathypelagic foraging zones, holding eDNA of 39 cephalopod taxa, including 22 known prey. Contrary to expectation, extensive taxonomic overlap in prey spectra between foraging zones indicated that predator niche segregation was not driven by prey community composition alone. Instead, intraspecific prey spectrum differences may drive differentiation for hunting fewer, more calorific, mature cephalopods in deeper waters. The novel combination of methods presented here holds great promise to disclose elusive deep-sea predator-prey systems, aiding in their protection.

In situ observations show vertical community structure of pelagic fauna in the eastern tropical North Atlantic off Cape Verde.

Distribution patterns of fragile gelatinous fauna in the open ocean remain scarcely documented. Using epi-and mesopelagic video transects in the eastern tropical North Atlantic, which features a mild but intensifying midwater oxygen minimum zone (OMZ), we established one of the first regional observations of diversity and abundance of large gelatinous zooplankton. We quantified the day and night vertical distribution of 46 taxa in relation to environmental conditions. While distribution may be driven by multiple factors, abundance peaks of individual taxa were observed in the OMZ core, both above and below the OMZ, only above, or only below the OMZ whereas some taxa did not have an obvious distribution pattern. In the eastern eropical North Atlantic, OMZ expansion in the course of global climate change may detrimentally impact taxa that avoid low oxygen concentrations (Beroe, doliolids), but favour taxa that occur in the OMZ (Lilyopsis, phaeodarians, Cydippida, Colobonema, Haliscera conica and Halitrephes) as their habitat volume might increase. While future efforts need to focus on physiology and taxonomy of pelagic fauna in the study region, our study presents biodiversity and distribution data for the regional epi- and mesopelagic zones of Cape Verde providing a regional baseline to monitor how climate change may impact the largest habitat on the planet, the deep pelagic realm.

Trace element analysis reveals bioaccumulation in the squid Gonatus fabricii from polar regions of the Atlantic Ocean.

The boreoatlantic gonate squid (Gonatus fabricii) represents important prey for top predators-such as marine mammals, seabirds and fish-and is also an efficient predator of crustaceans and fish. Gonatus fabricii is the most abundant cephalopod in the northern Atlantic and Arctic Ocean but the trace element accumulation of this ecologically important species is unknown. In this study, trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn) were analysed from the mantle muscle and the digestive gland tissue of juveniles, adult females, and adult males that were captured south of Disko Island off West-Greenland. To assess the feeding habitat and trophic position of this species, stable isotopes of carbon (δ13C) and nitrogen (δ15N) were measured in their muscle tissue. Mercury concentrations were positively correlated with size (mantle length) and trophic position. The Hg/Se ratio was assessed because Se has been suggested to play a protective role against Hg toxicity and showed a molar surplus of Se relative to Hg. Cadmium concentrations in the digestive gland were negatively correlated with size and trophic position (δ15N), which suggested a dietary shift from Cd-rich crustaceans towards Cd-poor fish during ontogeny. This study provides trace element concentration data for G. fabricii from Greenlandic waters, which represents baseline data for a northern cephalopod species. Within West-Greenland waters, G. fabricii appears to be an important vector for the transfer of Cd in the Arctic pelagic food web.

High cadmium and mercury concentrations in the tissues of the orange-back flying squid, Sthenoteuthis pteropus, from the tropical Eastern Atlantic.

The orange-back flying squid, Sthenoteuthis pteropus, plays an important role in the eastern tropical Atlantic Ocean (ETA) pelagic food web, as both predator and prey. Specimens of S. pteropus were caught off the Cape Verde Islands and concentrations of Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V, and Zn were measured in the digestive gland. Among the analysed elements, Cd showed the highest average concentration with values among the highest ever recorded in cephalopods. In addition to the digestive gland, Hg concentrations were also analysed in the buccal mass and mantle tissue. Among the three tissues, buccal mass showed the highest Hg concentrations. In females, Hg concentrations in the buccal mass were positively correlated with stable isotope ratios (δ13C and δ15N) and mantle length, showing both bioaccumulation with age and bioamplification along the trophic levels. High Cd and Hg concentrations in the digestive gland and muscle respectively would lead to elevated exposure of squid-eating top predators such as yellowfin tuna, swordfish or dolphinfish, which are commercially harvested for human consumption. This study provides a deeper understanding of the trace element contamination in an abundant and ecologically important, but poorly studied pelagic squid in the ETA.

Bathyal feasting: post-spawning squid as a source of carbon for deep-sea benthic communities.

In many oceanic carbon budgets there is a discrepancy between the energetic requirements of deep-sea benthic communities and the supply of organic matter. This suggests that there are unidentified and unmeasured food sources reaching the seafloor. During 11 deep-sea remotely operated vehicle (ROV) surveys in the Gulf of California, the remains (squid carcasses and hatched-out egg sheets) of 64 post-brooding squid were encountered. As many as 36 remains were encountered during a single dive. To our knowledge this is one of the largest numbers of natural food falls of medium-size deep-sea nekton described to date. Various deep-sea scavengers (Ophiuroidea, Holothuroidea, Decapoda, Asteroidea, Enteropneusta) were associated with the remains. Although many of the 80 examined ROV dives did not encounter dead squids or egg sheets (n = 69), and the phenomenon may be geographically and temporally restricted, our results show that dead, sinking squid transport carbon from the water column to the seafloor in the Gulf of California. Based on food fall observations from individual dives, we estimate that annual squid carcass depositions may regionally contribute from 0.05 to 12.07 mg C m-2 d-1 to the seafloor in the areas where we observed the remains. The sinking of squid carcasses may constitute a significant but underestimated carbon vector between the water column and the seafloor worldwide, because squid populations are enormous and are regionally expanding as a result of climate change and pressure on fish stocks. In the future, standardized methods and surveys in geographical regions that have large squid populations will be important for investigating the overall contribution of squid falls to regional carbon budgets.

The giant deep-sea octopus Haliphron atlanticus forages on gelatinous fauna.

Feeding strategies and predator-prey interactions of many deep-sea pelagic organisms are still unknown. This is also true for pelagic cephalopods, some of which are very abundant in oceanic ecosystems and which are known for their elaborate behaviors and central role in many foodwebs. We report on the first observations of the giant deep-sea octopus Haliphron atlanticus with prey. Using remotely operated vehicles, we saw these giant octopods holding medusae in their arms. One of the medusae could be identified as Phacellophora camtschatica (the egg-yolk jelly). Stomach content analysis confirmed predation on cnidarians and gelatinous organisms. The relationship between medusae and H. atlanticus is discussed, also in comparison with other species of the Argonautoidea, all of which have close relationships with gelatinous zooplankton.

Preclinical evaluation of a novel ¹¹¹In-labeled bombesin homodimer for improved imaging of GRPR-positive prostate cancer.

Rational-designed multimerization of targeting ligands can be used to improve kinetic and thermodynamic properties. Multimeric targeting ligands may be produced by tethering multiple identical or two or more monomeric ligands of different binding specificities. Consequently, multimeric ligands may simultaneously bind to multiple receptor molecules. Previously, multimerization has been successfully applied on radiolabeled RGD peptides, which resulted in an improved tumor targeting activity in animal models. Multimerization of peptide-based ligands may improve the binding characteristics by increasing local ligand concentration and by improving dissociation kinetics. Here, we present a preclinical study on a novel radiolabeled bombesin (BN) homodimer, designated (111)In-DOTA-[(Aca-BN(7-14)]2, that was designed for enhanced targeting of gastrin-releasing peptide receptor (GRPR)-positive prostate cancer cells. A BN homodimer was conjugated with DOTA-NHS and labeled with (111)In. After HPLC purification, the GRPR targeting ability of (111)In-DOTA-[Aca-BN(7-14)]2 was assessed by microSPECT imaging in SCID mice xenografted with the human prostate cancer cell line PC-3. (111)In labeling of DOTA-[(Aca-BN(7-14)]2 was achieved within 30 min at 85 °C with a labeling yield of >40%. High radiochemical purity (>95%) was achieved by HPLC purification. (111)InDOTA-[Aca-BN(7-14)]2 specifically bound to GRPR-positive PC-3 prostate cancer cells with favorable binding characteristics because uptake of 111In-DOTA-[Aca-BN(7-14)]2 in GRPR-positive PC-3 cells increased over time. A maximum peak with 30% radioactivity was observed after 2 h of incubation. The log D value was -1.8 ± 0.1. (111)In-DOTA-[Aca-BN(7-14)]2 was stable in vitro both in PBS and human serum for at least 4 days. In vivo biodistribution analysis and microSPECT/CT scans performed after 1, 4, and 24 h of injection showed favorable binding characteristics and tumor-to-normal tissue ratios. This study identifies (111)In-DOTA-[(Aca-BN(7-14)]2 as a promising radiotracer for nuclear imaging of GRPR in prostate cancer.

Mating behavior of a deep-sea squid revealed by in situ videography and the study of archived specimens.

The mating behavior of deep-sea squids is shrouded in mystery. The squids for which mating has been observed use a hectocotylus, a modified arm, for the transfer of sperm packets called spermatophores. However, many deep-sea squid species lack a hectocotylus. We present the first in situ observations of mating behavior in a deep-sea squid that has no hectocotylus but instead uses an elongated terminal organ for the transfer of spermatangia, which are released from the spermatophores and burrow deeply into the female tissue. With remotely operated vehicles (ROVs), we observed two mating pairs of the deep-sea squid Pholidoteuthis adami in the Gulf of Mexico. The male adopted a peculiar position during mating, with its ventral side up and its posterior mantle above the female's head. While the male held the female in what looked like a firm grip, we observed the long terminal organ extending through the funnel of the male, contacting the female dorsal mantle. Examinations of museum specimens show that spermatangia burrow from the outer dorsal mantle into the inner dorsal mantle. This combination of serendipitous in situ observations and archived specimens can be a powerful tool for understanding the behavior of deep-sea animals.

Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I.

The stereochemistry of the transcarboxylase-catalyzed carboxylation of 3-fluoropyruvate has been studied by using fluorine NMR of unpurified reaction mixtures. When the product 3-fluorooxaloacetate was trapped by using malate dehydrogenase, only the 2R,3R diastereomer of 3-fluoromalate was formed. The fluoromethyl group of fluoropyruvate does not take up deuterium label from the solvent during the reaction. These results confirm and extend those obtained previously by Walsh and co-workers [Goldstein, J. A., Cheung, Y. F., Marletta, M. A., & Walsh, C. (1978) Biochemistry 17, 5567-5575] showing that transcarboxylase is specific for one of the two prochiral hydrogens in fluoropyruvate. Transcarboxylase, coupled to malate dehydrogenase, has been used to analyze samples of chiral fluoropyruvate obtained by dephosphorylation of (Z)-fluorophosphoenolpyruvate in D2O in the presence of either pyruvate kinase or enzyme I from the Escherichia coli sugar transport systems. Analysis of the fluoromalate produced showed that fluoroenolpyruvate is deuterated from opposite faces by these two enzymes: enzyme I protonates (deuterates) fluoroenolpyruvate exclusively from the 2-re face and pyruvate kinase does so mainly from the 2-si face. Fluoropyruvate is carboxylated by transcarboxylase with absolute retention of configuration.

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: stereospecificity of proton transfer in the phosphorylation of enzyme I from (Z)-phosphoenolbutyrate.

The stereochemistry of the proton transfer in the reaction of phosphoenolbutyrate with enzyme I has been established. During the reaction of the pure Z isomer of this analogue of phosphoenolpyruvate with enzyme I, to yield phosphoenzyme I and 2-oxobutyrate, the substrate is protonated at C-3 from the 2re,3si face. This stereospecificity was established for the transfer of a proton to (Z)-phospho[3-D]enolbutyrate and for the transfer of a deuteron to (Z)-phospho[3-H]enolbutyrate. The E isomer of phosphoenolbutyrate is not a substrate for enzyme I. Accordingly, the reaction of phosphoenzyme I with 2-oxobutyrate yields exclusively the Z isomer of phosphoenolbutyrate, and only the pro-S proton at C-3 of 2-oxobutyrate is abstracted. A kinetic H/D isotope effect of 6.8 in this reaction demonstrates the rate-limiting nature of the proton-transfer step. The stereochemical analysis of 2-oxo[3(R)-H,D]butyrate and of 2-oxo-[3(S)-H,D]butyrate was carried out by using the pyruvate kinase catalyzed enolization of this compound. This enzymatic enolization, with phosphate as a cofactor, is rapid at neutral pH and is a highly stereospecific reaction: only the pro-R proton at C-3 of 2-oxobutyrate is exchanged with solvent. This reaction was also used to generate the pure 3R and 3S enantiomers of 2-oxo[3-H,D]butyrate. The degree of protonation/deuteration at C-3 of 2-oxobutyrate was detected from the fine structure of the methyl proton nuclear magnetic resonance signal.

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: role of divalent metals in the dimerization and phosphorylation of enzyme I.

The function of divalent metal ions (Mg2+ and Mn2+) in the dimerization and phosphorylation of enzyme I has been studied. Only a dimeric form of the enzyme can be phosphorylated [Misset, O., Brouwer, M., & Robillard, G. T. (1980) Biochemistry 19, 883--890; Hoving, H., Lolkema, J. S., & Robillard, G. T. (1981) Biochemistry 20, 87--93]. Kinetic studies of phosphoryl-group exchange between phosphoenolpyruvate and pyruvate and measurements of initial enzyme I phosphorylation rates revealed that a divalent metal ion must be bound to the enzyme to render the dimer active. Mn2+ binding experiments by means of electron paramagnetic resonance showed binding of at least one Mn2+ per unphosphorylated dimer with a binding constant comparable to the activation constant found in the kinetic studies and a 10-fold tighter binding of only one Mn2+ per phosphorylated dimer. Gel filtration experiments provided evidence that divalent metals produce about a 10-fold stabilization of the dimers, in addition to their effect on the specific dimer activity. The stability of the dimer was also strongly dependent on salts such as LiCl, NaCl, KCl, and a series of tetraalkylammonium chlorides. The relative effects of these salts suggest that hydrophobic interactions possibly play a significant role in enzyme I dimerization.

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: equilibrium kinetics and mechanism of enzyme i phosphorylation.

The phosphorylation of enzyme I from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system was studied by means of isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H--2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate. 14C--12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect (nu V/nu D = 1.9), showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer. Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated. From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse second-order rate constants of the reaction were determined to be 3 X 10(7) and 8 X 10(5) M-1 min-1, respectively, yielding an equilibrium constant of approximately 40 and a delta G degree for enzyme I phosphorylation of --2.3 kcal/mol. The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.

An infrared study of carbon monoxide complexes of hemocyanins. Evidence for the structure of the co-binding site from vibrational analysis.

A vibrational analysis was carried out showing that the infrared experimental data of 13C and 18O carbon monoxide complexes of hemocyanin of Fager and Alben (Biochemistry 11 (1972) 4786) are consistent with a coordination of the carbon atom of CO to one of the two copper ions in the active site. This conclusion contradicts the original interpretation of Fager and Alben in which oxygen-coordination to copper was suggested. This vibrational analysis can also be applied to the study of Alben and Caughey (Biochemistry 7 (1968) 175) with 13C and 18O carbonyl hemoglobin, in which oxygen-coordination to iron was suggested. Carbonyl hemocyanins from several sources have also been studied by infrared spectroscopy. The single stretching vibration of CO bound to arthropodal (Cancer magister) hernocyanin (nu(co)) is at 2042.5 cm(-1), while nu(CO) for gastropod (Helix pomatia of the phylum Mollusca) alpha and beta hemocyanin is at 2064.5 cm(-1)and 2062.5 cm(-1), respectively. The intensities of the CO stretching bands were all around 1.5 X 10(4) M(-1) cm(-2). Calculations show that with the present attainable accuracy it is impossible to detect hydrogen bonding of exchangeable protons to small molecules bound to proteins (for example CO), by comparing its stretching frequencies in H2O and D2O buffers.

Transcriptional units for ribosomal proteins in yeast.

The effects of ultraviolet irradiation on the rates of synthesis of individual ribosomal proteins in yeast were examined and compared with the ultraviolet sensitivities of the synthesis of other yeast proteins. It was found that the synthesis of yeast ribosomal proteins is much more sensitive to ultraviolet irradiation than that of other yeast cellular proteins. Taking into account the half-life of yeast mRNA, the results obtained indicate that the genes coding for ribosomal proteins form part of long transcriptional units, which are much longer than the DNA stretch needed to code for a ribosomal protein of average molecular weight. Saturation hybridization of total poly(A)-containing mRNA with yeast nuclear DNA revealed that as much as 30% of DNA is complementary to yeast mRNA. Thus, the primary transcript of a protein gene on the average is about 1.7 times the length of the actual messenger. On the basis of the various experimental data we suggest a clustering of the yeast ribosomal protein genes in a number of common transcriptional units.

Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site.

The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.