An investigation of the mechanisms for sterol synthesis and dietary sterol bioconversion in the heterotrophic protists Oxyrrhis marina and Gyrodinium dominans

The ability of the marine heterotrophic protists Oxyrrhis marina and Gyrodinium dominans to synthesize sterols de novo and modify dietary sterols was investigated using ¹³C-labeled substrates. De novo sterol synthesis of O. marina was determined by incorporation of ¹³C acetate into the culture medium. For G. dominans which has low tolerance of acetate, a protozoan prey Perkinsus marinus that cannot synthesize sterols, was cultured with ¹³C acetate then fed to G. dominans. Both heterotrophs utilized dietary ¹³C to synthesize fatty acids de novo, but not sterols. The ability of O. marina and G. dominans to alkylate, saturate, and desaturate dietary sterols was tested using P. marinus incorporated with ¹³C-labeled cholesterol as prey. O. marina did not modify the dietary ¹³C-cholesterol, but G. dominans produced 5 labeled sterols (brassicasterol, C28:1, and unknown C28, C29 and C30 sterols) indicating that G. dominans has the ability to desaturate and alkylate dietary cholesterol. The ability of O. marina and G. dominans to dealkylate dietary sterols was tested by feeding them gelatin acacia microspheres (GAMs) containing ¹³C-labeled brassicasterol. Neither heterotroph dealkylated brassicasterol to make cholesterol, but G. dominans alkylated and saturated brassicasterol to make 2 sterols (C29:1 and C30:0). The lack of dealkylation of brassicasterol by both protist species suggests problems with the substrate and/or delivery system since previous studies suggest that dealkylation of brassicasterol occurs when either species is fed algae containing this sterol.     

Effect of dietary fatty acids on the body tissues of larval and juvenile cobia and their prey

Polyunsaturated fatty acids (PUFAs) have been used as biomarkers in pelagic ecosystems although previous studies have failed to quantify the timing of conservation of dietary PUFAs in pelagic fishes and invertebrates. Here we investigated the influence of diet upon the timing of conservation of PUFAs throughout multiple trophic exchanges in larval and juvenile cobia (Rachycentron canadum) and their prey. Cobia, rotifers (Brachionus plicatilis), and Artemia (A. franciscana) were fed laboratory processed or natural diets resembling prey and dietary modification of fatty acid signatures was quantified using two-source mixing models. Specimens were collected throughout the experiment to track dietary influences over time. Cobia larvae underwent significant dietary modification of PUFAs after 24 h and conserved > 90% of dietary PUFAs after an average of 6 days. Similar results were identified in juvenile cobia as significant dietary modification of PUFAs took place after 3 days and > 90% were conserved after an average of 12 days. In addition, no significant ontogenetic changes in PUFA signatures were identified in juvenile cobia throughout the 30-day experiment. PUFA signatures in prey items (rotifers and Artemia) underwent significant dietary modification in 24 h, with over 90% incorporation after 5-7 days. Results from this study support the premise that fatty acids are promising dietary indicators and may be useful for future studies examining trophic relationships in marine ecosystems and habitat use of marine fishes.     

Seasonal variations in bulk tissue, fatty acid and monosaccharide δC values of leaves from mesotrophic grassland plant communities under different grazing managements

Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ¹³C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ¹³C values could be determined. The total mean leaf bulk δ¹³C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ¹³C values was only determined in October (P = <0.001) when δ¹³C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields (P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ¹³C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ¹³C values had a major influence on bulk δ¹³C values. An average depletion of ca. 1‰ in the bulk δ¹³C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ¹³C values, as glucose constituted >50% total leaf monosaccharides. In October, δ¹³C values of all monosaccharides varied between species, with significant variation in δ¹³C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ¹³C values observed in October 1999. The fatty acids C_16:0, C_18:2 and C_18:3 were highly abundant in all plant species. Fatty acid δ¹³C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C_16:0), −37.0‰ (C_18:2) and −36.5‰ (C_18:3) were determined. There was significant interspecific variation in the δ¹³C values of all individual fatty acids during October and July, but only for C_18:2 in May (P = <0.05). This indicated that seasonal trends observed in the δ¹³C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ¹³C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends (P = <0.0001) for fatty acid and monosaccharide δ¹³C values: δ¹³C_16:0 < δ¹³C_18:2 < δ¹³C_18:3 and δ¹³C_arabinose > δ¹³C_xylose > δ¹³C_glucose > δ¹³C_galactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.     

Qualitative assessment of the diet of European eel larvae in the Sargasso Sea resolved by DNA barcoding

European eels (Anguilla anguilla) undertake spawning migrations of more than 5000 km from continental Europe and North Africa to frontal zones in the Sargasso Sea. Subsequently, the larval offspring are advected by large-scale eastward ocean currents towards continental waters. However, the Sargasso Sea is oligotrophic, with generally low plankton biomass, and the feeding biology of eel larvae has so far remained a mystery, hampering understanding of this peculiar life history. DNA barcoding of gut contents of 61 genetically identified A. anguilla larvae caught in the Sargasso Sea showed that even the smallest larvae feed on a striking variety of plankton organisms, and that gelatinous zooplankton is of fundamental dietary importance. Hence, the specific plankton composition seems essential for eel larval feeding and growth, suggesting a linkage between eel survival and regional plankton productivity. These novel insights into the prey of Atlantic eels may furthermore facilitate eel larval rearing in aquaculture, which ultimately may replace the unsustainable use of wild-caught glass eels.     

Determination of the toxicity of faeces of cattle treated with an ivermectin sustained-release bolus and preference trials using a dung fly, Neomyia cornicina

The toxicity of dung from cattle treated with an ivermectin sustained-release bolus was estimated in terms of ivermectin or ivermectin equivalents, using a laboratory bioassay with the dung fly Neomyia cornicina Fabricius (Diptera, Muscidae). The mortalities of flies measured 7 days after feeding for 24 h on dung containing known concentrations of ivermectin (between 0.125 and 1 g ivermectin per gram fresh dung) were compared with the mortalities of insects fed for 24 h on dung from cattle treated 21 days previously with an ivermectin sustained-release bolus. The toxicity of the bolus dung was equivalent to dung containing 0.66 g ivermectin per gram fresh dung. To determine whether insects could differentiate between control dung and dung from bolus-treated cattle, choice-chamber tests were carried out. There was no significant difference in the percentage of females that chose either dung type, suggesting that they were unable to distinguish the dung of bolus-treated cattle from control dung. Results are discussed in relation to the impact that bolus use can have on the insect fauna of cattle dung.     

Cloning,expression, and purification of a new antimicrobial peptide gene from Musca domestica larva

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram − bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.     

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction¹, boosting the immune response², pheromone production³, as well as nutrition, including the synthesis of essential amino acids^4, among others.    Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing ¹³C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA⁵. The incorporation of ¹³C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (¹²C) one. In the end, the ¹³C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the ¹²C-unlabeled similar one⁶. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, ¹³C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.     

Toward the physiological basis for increased Agrotis ipsilon multiple nucleopolyhedrovirus infection following feeding of Agrotis ipsilon larvae on transgenic corn expressing Cry1Fa2

Larvae of the black cutworm, Agrotis ipsilon Hufnagel, were more susceptible to infection by A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV: Baculoviridae) after feeding on Herculex^® I, a transgenic corn hybrid expressing the Bacillus thuringiensis (Bt)-derived toxin Cry1Fa2 compared to larvae fed on isoline corn. We investigated the physiological basis for increased susceptibility to virus infection following exposure to Herculex^® I by analyzing the midgut pH, gut protease activity and peritrophic matrix structure which are important factors for both Bt toxin action and baculovirus infection. No significant treatment differences were found in the pH of anterior midgut, central midgut or posterior midgut in larvae fed Herculex^® I or isoline diets. Analysis of soluble and membrane-associated gut proteinase activities from larvae fed Herculex^® I or isoline diets indicated that membrane-associated aminopeptidase activity and soluble chymotrypsin-like proteinase activity were significantly lower in Herculex^® I -fed larvae compared to isoline-fed larvae. The number and relative molecular masses of soluble chymotrypsin-like proteinases did not differ. Baculoviruses were not susceptible to in vitro degradation by bovine chymotrypsin, suggesting that chymotrypsin degradation of baculovirus occlusion-derived virus did not result in reduced infection of larvae fed on isoline diet. Scanning electron micrographs of the peritrophic matrices of Herculex^® I -fed larvae and isoline-fed larvae indicated that Herculex^® I did not result in damage to the peritrophic matrix that could facilitate subsequent baculovirus infection. Additional research is required to further delineate the physiological basis for enhanced baculovirus infection following exposure to sublethal doses of Bt toxins.     

Rapidly increasing methyl mercury in endangered ivory gull (Pagophila eburnea) feathers over a 130 year record

Mercury (Hg) is increasing in marine food webs, especially at high latitudes. The bioaccumulation and biomagnification of methyl mercury (MeHg) has serious effects on wildlife, and is most evident in apex predators. The MeHg body burden in birds is the balance of ingestion and excretion, and MeHg in feathers is an effective indicator of overall MeHg burden. Ivory gulls (Pagophila eburnea), which consume ice-associated prey and scavenge marine mammal carcasses, have the highest egg Hg concentrations of any Arctic bird, and the species has declined by more than 80% since the 1980s in Canada. We used feathers from museum specimens from the Canadian Arctic and western Greenland to assess whether exposure to MeHg by ivory gulls increased from 1877 to 2007. Based on constant feather stable-isotope (δ¹³C, δ¹⁵N) values, there was no significant change in ivory gulls'' diet over this period, but feather MeHg concentrations increased 45× (from 0.09 to 4.11 µg g⁻¹ in adults). This dramatic change in the absence of a dietary shift is clear evidence of the impact of anthropogenic Hg on this high-latitude threatened species. Bioavailable Hg is expected to increase in the Arctic, raising concern for continued population declines in high-latitude species that are far from sources of environmental contaminants.     

Assimilation and biosynthesis of lipids in Arctic Calanus species based on feeding experiments with a C labelled diatom

The dominant Arctic Ocean and North Atlantic copepods Calanus hyperboreus, Calanus glacialis, and Calanus finmarchicus were collected in the Greenland Sea and fed ¹³C labelled diatom Thalassiosira weissflogii to follow the transfer and assimilation of carbon, lipid, and individual fatty acids and alcohols. The diatom was grown with ¹³C for 3 to 5 days and fed then to the copepods. During the feeding period of 14 days, total carbon increased in the copepodite stages V of C. hyperboreus and C. finmarchicus, whereas carbon remained almost constant in C. glacialis females. However, total lipid increased in all species and stages. Highest lipid accumulation occurred in C. hyperboreus in which nearly all lipids were exchanged already after 11 days of feeding. In the other species lipid accumulation made up between 22% (C. finmarchicus) and 45% of total lipid (C. glacialis). The proportion of wax esters was high ranging from 76% of total lipid in C. glacialis to 92% in C. finmarchicus. The fatty acid composition of the alga was dominated by 16:1(n-7), 16:0, 20:5(n-3), and 22:6(n-3). The composition of the copepods was similar because of feeding already on diatoms in the field. In addition, the monounsaturated fatty acids and alcohols, 20:1(n-9) and 22:1(n-11), were major components of the copepod lipids. During the feeding period the highest ¹³C labelling was always found in the C₁₆ polyunsaturated fatty acids and in the 16:1(n-7) alcohol. Because these components occurred only in trace amounts in the copepods they totally originated from the diet explaining the high labelling. It is noteworthy that the 16:1(n-7) alcohol originated only from the corresponding dietary and not from the abundant internal fatty acid. The long-chain monounsaturated fatty acids and alcohols, 20:1(n-9) and 22:1(n-11), are not existent in phytoplankton and have to be produced de novo. They were less labelled in the smaller species but highly ¹³C enriched in C. hyperboreus. Although dietary fatty acids were generally retained by the copepods it seems that fatty acids or even lipids were selectively accumulated and turned over due to bodily requirements, and thus, essential polyunsaturated fatty acids were preferentially retained. During feeding mixing, accumulation, and exchange of internal and dietary fatty acids and alcohols occurred as well as utilisation of lipids from both sources for metabolic requirements. The differences in lipid assimilation fit to the different life strategies of the copepods.     

The effect of the dung pad fauna on the emergence ofMusca tempestiva [Dipt.: Muscidae] from dung pads in southern France

The emergence ofMusca tempestiva Fallèn, from cow pads in southern France was reduced by a mean of 98% and 96% respectively in nine experiments concluded in 1979 and 1981, by the action of the complete dung fauna. Fauna emerging from dung pads and captured in simultaneous trapping using dung baited pitfall traps consisted of 13 Dipterous species (9 Muscidae, 2 Sepsidae and 2 Sphaeroceridae), 25 Coleopterous species (7 Scarabaeidae, 1 Geotrupidae, 2 Aphodiidae, 3 Hydrophilidae, 12 Staphylinidae), 3 Hymenopterous parasites and 1 species of macrochelid and 2 species of parasitoid mites carried phoretically by the dung beetles. The total number of insects and mites per trap was usually small. The Staphylinidae which included 4Aleochara species of whichAleochara tristits Gravenhorst andA. bipustulata (L.) are known parasitoids of Diptera pupae, and 6 predatory species, were the most abundant insects present. Whilst the overall abundance of insects and mites trapped was relatively low it is probable that the most important factor in the control ofM. tempestiva was the combined influence of the different elements making up the dung pad fauna.     

Immune Correlates of Resistance to Trichinella spiralis Reinfection in Mice

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23⁺ IgM⁺ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23⁺ IgM⁺ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23⁺ IgM⁺ B cell responses.     

Capture of algae promotes growth and propagation in aquatic Utricularia

Background and Aims Some carnivorous plants trap not only small animals but also algae and pollen grains. However, it remains unclear if these trapped particles are useless bycatch or whether they provide nutrients for the plant. The present study examines this question in Utricularia, which forms the largest and most widely spread genus of carnivorous plants, and which captures prey by means of sophisticated suction traps.Methods Utricularia plants of three different species (U. australis, U. vulgaris and U. minor) were collected in eight different water bodies including peat bogs, lakes and artificial ponds in three regions of Austria. The prey spectrum of each population was analysed qualitatively and quantitatively, and correlated with data on growth and propagation, C/N ratio and δ¹⁵N.Key Results More than 50 % of the prey of the Utricularia populations investigated consisted of algae and pollen, and U. vulgaris in particular was found to capture large amounts of gymnosperm pollen. The capture of algae and pollen grains was strongly correlated with most growth parameters, including weight, length, budding and elongation of internodes. The C/N ratio, however, was less well correlated. Other prey, such as moss leaflets, fungal hyphae and mineral particles, were negatively correlated with most growth parameters. δ¹⁵N was positively correlated with prey capture, but in situations where algae were the main prey objects it was found that the standard formula for calculation of prey-derived N was no longer applicable.Conclusions The mass capture of immotile particles confirms the ecological importance of autonomous firing of the traps. Although the C/N ratio was little influenced by algae, they clearly provide other nutrients, possibly including phosphorus and trace elements. By contrast, mosses, fungi and mineral particles appear to be useless bycatch. Correlations with chemical parameters indicate that Utricularia benefits from nutrient-rich waters by uptake of inorganic nutrients from the water, by the production of more traps per unit of shoot length, and by the capture of more prey particles per trap, as nutrient-rich waters harbour more prey organisms.     

Interactions between fungi and bacteria influence microbial community structure in the Megachile rotundata larval gut

Recent declines in bee populations coupled with advances in DNA-sequencing technology have sparked a renaissance in studies of bee-associated microbes. Megachile rotundata is an important field crop pollinator, but is stricken by chalkbrood, a disease caused by the fungus Ascosphaera aggregata. To test the hypothesis that some gut microbes directly or indirectly affect the growth of others, we applied four treatments to the pollen provisions of M. rotundata eggs and young larvae: antibacterials, antifungals, A. aggregata spores and a no-treatment control. We allowed the larvae to develop, and then used 454 pyrosequencing and quantitative PCR (for A. aggregata) to investigate fungal and bacterial communities in the larval gut. Antifungals lowered A. aggregata abundance but increased the diversity of surviving fungi. This suggests that A. aggregata inhibits the growth of other fungi in the gut through chemical or competitive interaction. Bacterial richness decreased under the antifungal treatment, suggesting that changes in the fungal community caused changes in the bacterial community. We found no evidence that bacteria affect fungal communities. Lactobacillus kunkeei clade bacteria were common members of the larval gut microbiota and exhibited antibiotic resistance. Further research is needed to determine the effect of gut microbes on M. rotundata health.     

Effect of lipid removal on carbon and nitrogen stable isotope ratios in crustacean tissues

The analysis of tissue's naturally occurring stable carbon and nitrogen isotope ratios is a useful tool to delineate trophic relationships. However, the interpretation of δ¹³C and δ¹⁵N is complicated by the influence of multiple factors such as the tissue-specific lipid content. The aim of this work was to evaluate the effects of lipid extraction on δ¹³C and δ¹⁵N compositions in muscle, hepatopancreas and gonads of a marine decapod crustacean, the spider crab Maja brachydactyla. Samples were analyzed for stable isotopes before and after lipid removal, using a derived Soxhlet extraction method. Differences in δ¹³C and δ¹⁵N were measured among tissues before and after treatment. Lipid extraction of muscle did not have a significant effect on either δ¹³C or δ¹⁵N. By contrast, ecologically significant shifts for both carbon and nitrogen stable isotopes ratios (+ 2.9 ± 0.8‰ for δ¹³C, and + 1.2 ± 0.7‰ for δ¹⁵N) were noticed in the hepatopancreas. In regard to gonads, lipid extraction led to a shift only on δ¹³C (+ 1.3 ± 0.3‰). Finally, the derived Soxhlet extraction method removed the lipid influence for δ¹³C, and had an effect on δ¹⁵N composition for lipid-rich samples. We recommend this treatment for carbon stable isotope studies on decapod crustacean lipid-rich tissues.     

Red porgy (Pagrus pagrus) larval feeding performance and behavior at the onset of exogenous feeding

The feeding performance and behavior at the onset of exogenous feeding, 3 to 4 days after hatching (DAH), were studied in red porgy Pagrus pagrus larvae. Similar feeding efficiency and intensity were achieved for two feeding treatments (live or freeze-dried rotifers) suggesting that prey movement is not decisive for their detection and capture and demonstrating that at first feeding red porgy larvae can ingest inert food. Larvae feeding performance was not affected by a diet shift between treatments. Based on maximum rotifers consumption and gut evacuation time at 18 °C, the daily ration was estimated as 14.035 μg, considering 14 h of feeding and a 25% egg:female rotifer ratio. Larval swimming activity measured by video recording showed a close association with gut fullness and similar swimming patterns for 3 and 4 DAH larvae. However, 20.3% larger mouth gape and 54.6% higher swimming speed of the older larvae should provide a better feeding performance and more energy needed for growth.     

Weak meiofaunal trophic linkages in Crangon crangon and Carcinus maenus

To document the relative importance of meiofauna as prey for juvenile Crangon crangon and Carcinus maenus, short interval (1.5-2 h) collections were made in the muddy Lynher Estuary (Plymouth, Great Britain) and in the sandy-bottom Ythan Estuary (Aberdeenshire, Scotland) in 1990. Gut passage times of Crangon fed flaked fish food and fluorescent tracer in the laboratory at 13 °C ranged from 4 to 20 h. Wild shrimp exhibited feeding periodicity, with guts fullest during high tide in both locations. Visual and immunological gut contents analyses revealed that meiofaunal nematodes and harpacticoid copepods were present only in recently settled shrimp from 8 to 12 mm total length on muddy bottoms. Larger shrimp collectively consumed up to 33 different macrobenthic prey types. Shrimp were fullest at night (mean gut contents weight = 8% wet body weight, Lynher) or at dawn (6%, Ythan). The Lynher Carcinus gut contents—from animals 8 to 30 mm carapace width, examined visually only—contained mostly fluids, green benthic algae, sediment particles, and masses of unidentifiable prey remains plus digestion-resistant hard parts visually identifiable as macrobenthic in origin. None of the 203 crabs examined from the 24-h collection contained meiofaunal prey. Crangon shrimp probably eat meiofaunal prey for only a brief period of time after their initial settlement to the bottom. Evidence for significant top-down impacts on meiofauna from these two abundant shallow-water predators was weak. More trophic studies are needed on newly settled epibenthic predators to test the hypothesis that biological control of shallow-water meiofauna is important.     

Refined Analysis of Brain Energy Metabolism Using In Vivo Dynamic Enrichment of 13C Multiplets

Carbon-13 nuclear magnetic resonance spectroscopy in combination with the infusion of ¹³C-labeled precursors is a unique approach to study in vivo brain energy metabolism. Incorporating the maximum information available from in vivo localized ¹³C spectra is of importance to get broader knowledge on cerebral metabolic pathways. Metabolic rates can be quantitatively determined from the rate of ¹³C incorporation into amino acid neurotransmitters such as glutamate and glutamine using suitable mathematical models. The time course of multiplets arising from ¹³C-¹³C coupling between adjacent carbon atoms was expected to provide additional information for metabolic modeling leading to potential improvements in the estimation of metabolic parameters.The aim of the present study was to extend two-compartment neuronal/glial modeling to include dynamics of ¹³C isotopomers available from fine structure multiplets in ¹³C spectra of glutamate and glutamine measured in vivo in rats brain at 14.1 T, termed bonded cumomer approach. Incorporating the labeling time courses of ¹³C multiplets of glutamate and glutamine resulted in elevated precision of the estimated fluxes in rat brain as well as reduced correlations between them.     

Functional evidence for physiological mechanisms to circumvent neurotoxicity of cardenolides in an adapted and a non-adapted hawk-moth species

Because cardenolides specifically inhibit the Na⁺K⁺-ATPase, insects feeding on cardenolide-containing plants need to circumvent this toxic effect. Some insects such as the monarch butterfly rely on target site insensitivity, yet other cardenolide-adapted lepidopterans such as the oleander hawk-moth, Daphnis nerii, possess highly sensitive Na⁺K⁺-ATPases. Nevertheless, larvae of this species and the related Manduca sexta are insensitive to injected cardenolides. By radioactive-binding assays with nerve cords of both species, we demonstrate that the perineurium surrounding the nervous tissue functions as a diffusion barrier for a polar cardenolide (ouabain). By contrast, for non-polar cardenolides such as digoxin an active efflux carrier limits the access to the nerve cord. This barrier can be abolished by metabolic inhibitors and by verapamil, a specific inhibitor of P-glycoproteins (PGPs). This supports that a PGP-like transporter is involved in the active cardenolide-barrier of the perineurium. Tissue specific RT-PCR demonstrated expression of three PGP-like genes in hornworm nerve cords, and immunohistochemistry further corroborated PGP expression in the perineurium. Our results thus suggest that the lepidopteran perineurium serves as a diffusion barrier for polar cardenolides and provides an active barrier for non-polar cardenolides. This may explain the high in vivo resistance to cardenolides observed in some lepidopteran larvae, despite their highly sensitive Na⁺K⁺-ATPases.     

Dietary conjugated linoleic acid alters long chain polyunsaturated fatty acid metabolism in brain and liver of neonatal pigs

Effects of dietary conjugated linoleic acid (CLA, 1% mixed isomers) on n-6 long-chain polyunsaturated fatty acid (LCPUFA) oxidation and biosynthesis were investigated in liver and brain tissues of neonatal piglets. Fatty acid β-oxidation was measured in tissue homogenates using [1-¹⁴C]linoleic acid (LA) and -arachidonic acid (ARA) substrates, while fatty acid desaturation and elongation were traced using [U-¹³C]LA and GC-MS. Dietary CLA had no effect on fatty acid β-oxidation, but significantly decreased n-6 LCPUFA biosynthesis by inhibition of LA elongation and desaturation. Differences were noted between our ¹³C tracer assessment of desaturation/elongation and simple precursor-product indices computed from fatty acid composition data, indicating that caution should be exercised when employing the later. The inhibitory effects of CLA on elongation/desaturation were more pronounced in pigs fed a low fat diet (3% fat) than a high fat diet (25% fat). Direct elongation of linoleic acid to C20:2n-6 via the alternate elongation pathway might play an important role in n-6 LCPUFA synthesis because more than 40% of the synthetic products of [U-¹³C]LA accumulated in [¹³C]20:2n-6. Overall, the data show that dietary CLA shifted the distribution of the synthetic products of [U-¹³C]LA between elongation and desaturation in liver and decreased the total synthetic products of [U-¹³C]LA in brain by inhibiting LA elongation to C20:2n-6. The impact of CLA on brain LCPUFA metabolism of the developing neonate merits consideration and further investigation.