Proteomic analysis of a sea-ice diatom: salinity acclimation provides new insight into the dimethylsulfoniopropionate production pathway

Dimethylsulfoniopropionate (DMSP) plays important roles in oceanic carbon and sulfur cycling and may significantly impact climate. It is a biomolecule synthesized from the methionine (Met) pathway and proposed to serve various physiological functions to aid in environmental stress adaptation through its compatible solute, cryoprotectant, and antioxidant properties. Yet, the enzymes and mechanisms regulating DMSP production are poorly understood. This study utilized a proteomics approach to investigate protein changes associated with salinity-induced DMSP increases in the model sea-ice diatom Fragilariopsis cylindrus (CCMP 1102). We hypothesized proteins associated with the Met-DMSP biosynthesis pathway would increase in relative abundance when challenged with elevated salinity. To test this hypothesis axenic log-phase cultures initially grown at a salinity of 35 were gradually shifted to a final salinity of 70 over a 24-h period. Intracellular DMSP was measured and two-dimensional gel electrophoresis was used to identify protein changes at 48 h after the shift. Intracellular DMSP increased by approximately 85% in the hypersaline cultures. One-third of the proteins increased under high salinity were associated with amino acid pathways. Three protein isoforms of S-adenosylhomo-cysteine hydrolase, which synthesizes a Met precursor, increased 1.8- to 2.1-fold, two isoforms of S-adenosyl Met synthetase increased 1.9- to 2.5-fold, and S-adenosyl Met methyltransferase increased by 2.8-fold, suggesting active methyl cycle proteins are recruited in the synthesis of DMSP. Proteins from the four enzyme classes of the proposed algal Met transaminase DMSP pathway were among the elevated proteins, supporting our hypothesis and providing candidate genes for future characterization studies.     

Characterization of microsatellite loci in sea urchins (Strongylocentrotus spp.)

We present six dinucleotide repeats that were developed and characterized in Strongylocentrotus droebachiensis and also tested in S. purpuratus. Four of these loci are polymorphic in S. droebachiensis (13–20 alleles, N= 100) and five are polymorphic in S. purpuratus (5–12 alleles, N= 10). We are currently using these markers to investigate the population substructure of shallow water populations of S. droebachiensis in the north Atlantic.     

Identification of the sea ice diatom biomarker IP25 in Arctic benthic macrofauna: direct evidence for a sea ice diatom diet in Arctic heterotrophs

Currently, the impact of declining seasonal sea ice extent in the Arctic on polar food webs remains uncertain. Previously, a range of proxy techniques has been employed to determine links between sea ice or phytoplankton primary production and the Arctic marine food web, although it is accepted that such approaches have their limitations. Here, we propose a novel approach to tracing sea ice primary production through Arctic food webs using the sea ice diatom biomarker, IP₂₅. Various benthic macrofaunal specimens were collected between March and May 2008 from Franklin Bay in the Amundsen Gulf, Arctic Canada, as part of the International Polar Year–Circumpolar Flaw Lead system study. Each specimen was analysed for the presence of the sea ice diatom biomarker IP₂₅ in order to provide evidence for feeding by benthic organisms on sea ice algae. IP₂₅ was found in nineteen out of the twenty-one specimens analysed, often as the most abundant of the highly branched isoprenoid biomarkers detected. The stable isotope composition of IP₂₅ (δ¹³C = −17.1 ± 0.5‰) in the sea urchin (Strongylocentrotus sp.) specimens was similar to that reported previously for this biomarker in Arctic sea ice, sedimenting particles and sediments. It is concluded that detection of IP₂₅ in Arctic benthic macrofauna represents a novel approach to providing convincing evidence for feeding on sea ice algae. It is also proposed that analysis of IP₂₅ may be used to trace trophic transfer of sea ice algal-derived organic matter through Arctic food webs in the future.     

Evidence for a cycloartenol-based sterol synthetic pathway inLagenidium spp.

Vegetative mycelia ofLagenidium giganteum andL. callinectes will metabolize cycloartenol to fucosterol and cholesterol. Lanosterol is not so metabolized although it is taken up from the culture broth. These data are indicative of a sterol synthetic pathway such as is present in photosynthetic organisms.     

The light-harvesting antenna of the diatom Phaeodactylum tricornutum. Evidence for a diadinoxanthin-binding subcomplex

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane-intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to establish whether different LHC subcomplexes exist, as well to determine an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. The major and more stable complex ( approximately 75% of total polypeptides) carries most of the chlorophyll a, and almost only one type of carotenoid, fucoxanthin. The minor complex, carrying approximately 10-15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition, suggesting specialized functions within the antenna. The diadinoxanthin-enriched complex could be where the de-epoxidation of diadinoxanthin into diatoxanthin mostly occurs.     

Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, alpha-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymotrypsin."     

Evidence for K+-dependent HCO3- utilization in the marine diatom Phaeodactylum tricornutum

Photosynthetic utilization of inorganic carbon in the marine diatom Phaeodactylum tricornutum was investigated by the pH drift experiment, measurement of K(1/2) values of dissolved inorganic carbon (DIC) with pH change, and comparison of the rate of photosynthesis with the rate of the theoretical CO(2) formation from uncatalyzed HCO(3)(-) conversion in the medium. The higher pH compensation point (10.3) and insensitivity of the photosynthetic rate to acetazolamide indicate that the alga has good capacity for direct HCO(3)(-) utilization. The photosynthetic rate reached 150 times the theoretical CO(2) supply rate at 100 micromol L(-1) DIC (pH 9.0) in the presence of 10 mmol L(-1) K(+) and 46 times that in the absence of K(+), indicating that for pH 9.4-grown P. tricornutum, HCO(3)(-) in the medium is taken up through K(+)-dependent and -independent HCO(3)(-) transporters. The K(1/2) (CO(2)) values at pH 8.2 were about 4 times higher than those at pH 9.0, whereas the K(1/2) (HCO(3)(-)) values at pH 8.2 were slightly lower than those at pH 9.0 whether without or with K(+), providing further evidence for the presence of the two HCO(3)(-) transport patterns in this alga. Photosynthetic rate and affinity for HCO(3)(-) in the presence of K(+), respectively, were about 2- and 7-fold higher than those in the absence of K(+), indicating that K(+)-dependent HCO(3)(-) transport is a predominant pattern of HCO(3)(-) cellular uptake in low DIC concentration. However, as P. tricornutum was cultured at pH 7.2 or 8.0, photosynthetic affinities to HCO(3)(-) were not affected by K(+), implying that K(+)-dependent HCO(3)(-) transport is induced when P. tricornutum is cultured at high alkaline pH.     

Some microbial contaminants and control agents in a diet and larvae of Heliothis spp

Sixteen tests were conducted with four antimicrobial agents that are routinely incorporated into lepidopterous larval diets for bacterial and fungal inhibition. The agents tested in all possible combinations were benomyl, chlortetracycline, sorbic acid, and methyl P-hydroxybenzoate. Those combinations that contained benomyl and sorbic acid in the formula were effective in complete microbial suppression during the larval portion of the 35-day testing period. Insect development in plastic cups of test diets with benomyl and sorbic acid averaged 97 and 93% pupation and adult emergence, respectively. The 12 tests that did not contain benomyl and sorbic acid were contaminated with some of the following fungi: Aspergillus niger, A. flavus, Rhizopus nigricans, Cladosporium, Fusarium, yeasts, or the bacterium, Bacillus subtilis. Microorganisms in Heliothis spp. larvae from mass-rearing diets were tested, examined, and identified. Bacteria recovered from laboratory-reared Heliothis larvae included Pseudomonas aeruginosa, P. maltophilia, Micrococcus luteus, α-hemolytic Streptococcus, Serratia marcescens, and S. rubidaea. Tests were conducted with sensi-discs for the selection of antimicrobials to arrest contamination in the larval digestive systems.     

Evidence of genetic variability for digestive efficiency in the growing pig fed a fibrous diet

Energy digestibility in the growing pig increases with BW increase and may differ between breeds of pigs or between lines selected on criteria other than digestion. However, little is known about the variability in energy digestibility within a line or a breed of pigs, especially when fibrous diets are fed. For this purpose, 20 Large White castrated male growing pigs originating from four boars (five per boar), and three to four sows per boar, were fed a high dietary fibre (DF) diet (18% NDF) and measured over 10 consecutive weeks (30 to 95 kg BW range) for their apparent faecal energy, nitrogen and organic matter digestibility. Each week, faeces were totally collected over 5 days and the feed dry matter intake over the same days was recorded. All digestibility coefficients increased regularly (P < 0.001) over the experimental periods or with BW increase (+0.6 point/10 kg BW increase for energy); this rate of increase was not affected by boar origin (no interaction; P > 0.05). The digestibility coefficients were affected by boar origin (P < 0.005 for energy), with about 2 points for energy between the extremes (81.7% v. 79.5%), and there was no marked interaction between boar origin and period. These preliminary results suggest the possibility of selecting growing pigs for an increased digestive efficiency when fed high DF diets.     

Lack of Evidence for Vertical Transmission of Campylobacter spp. in Chickens

Campylobacter jejuni is a major cause of bacterial food-borne infection in the industrial world. There is evidence that C. jejuni is present in eggs and hatchery fluff, opening the possibility for vertical transmission from hens to progeny. Poultry operations in Iceland provide an excellent opportunity to study this possibility, since breeding flocks are established solely from eggs imported from grandparent flocks in Sweden. This leaves limited opportunity for grandparents and their progeny to share isolates through horizontal transmission. While Campylobacter was not detected in all grandparent flocks, 13 of the 16 egg import lots consisted of eggs gathered from one or more Campylobacter-positive grandparent flocks. No evidence of Campylobacter was found by PCR in any of the 10 relevant quarantine hatchery fluff samples examined, and no Campylobacter was isolated from the parent birds through 8 weeks, while they were still in quarantine rearing facilities. After the birds were moved to less biosecure rearing facilities, Campylobacter was isolated, and 29 alleles were observed among the 224 isolates studied. While three alleles were found in both Sweden and Iceland, in no case was the same allele found both in a particular grandparent flock and in its progeny. We could find no evidence for vertical transmission of Campylobacter to the approximately 60,000 progeny parent breeders that were hatched from eggs coming from Campylobacter-positive grandparent flocks. If vertical transmission is occurring, it is not a significant source for the contamination of chicken flocks with Campylobacter spp.     

Evidence for paternal leakage in hybrid periodical cicadas (Hemiptera: Magicicada spp.)

Mitochondrial inheritance is generally assumed to be maternal. However, there is increasing evidence of exceptions to this rule, especially in hybrid crosses. In these cases, mitochondria are also inherited paternally, so "paternal leakage" of mitochondria occurs. It is important to understand these exceptions better, since they potentially complicate or invalidate studies that make use of mitochondrial markers. We surveyed F1 offspring of experimental hybrid crosses of the 17-year periodical cicadas Magicicada septendecim, M. septendecula, and M. cassini for the presence of paternal mitochondrial markers at various times during development (1-day eggs; 3-, 6-, 9-week eggs; 16-month old 1st and 2nd instar nymphs). We found evidence of paternal leakage in both reciprocal hybrid crosses in all of these samples. The relative difficulty of detecting paternal mtDNA in the youngest eggs and ease of detecting leakage in older eggs and in nymphs suggests that paternal mitochondria proliferate as the eggs develop. Our data support recent theoretical predictions that paternal leakage may be more common than previously estimated.     

The first evidence for genotypic stability in a cryopreserved transgenic diatom

Future algal biotechnology will need enhanced production strains, capable of more rapid growth, more efficient solar-energy conversion and/or higher levels of metabolite production. Almost certainly transgenic organisms will be used to ensure the cost-effective, economically viable production of a range of metabolites. As with all biotechnological processes, the functional stability, reliability and security of the production strains will be of paramount importance in algal biotechnology, as without this no biotechnological process is sustainable. In this study, the transgenic model strain Thalassiosira pseudonana CCAP 1085/23 was cryopreserved using a conventional, low-tech, colligative cryopreservation protocol. This employed dimethyl sulphoxide [5 % (v/v)] as a cryoprotectant, using a two-step cooling approach: initial controlled-rate cooling, followed by plunging into liquid nitrogen. High levels of post-thaw viability (70–85 %) were obtained, and on recovery of cryopreserved material no reduction in expression of the protein of the inserted gene (big1-GFP) was observed. Additionally, cryopreservation does not affect the localisation of the BIG1-GFP protein as demonstrated by microscopy of stained samples, nor its functionality as demonstrated by Western blotting.     

Methods for absolute diatom frequency analysis and combined diatom and pollen analysis in sediments

Earlier methods for absolute diatom frequency analysis were timeconsuming. In pollen analysis the use of indicator pollen and spores is well established. This method has been tested for absolute diatom analysis and proved to simplify the work. A detailed preparation procedure is given. A method for combined pollen and diatom analysis has been worked out to check the diatom production curves in sediments where the deposition rates can not be measured by ¹⁴C–dates or yearly laminated sediments. Diatom frequencies are here compared with synchronous terrestrial pollen frequencies. This gives an independent picture of changes in diatom productivity.     

三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建

三角褐指藻(Phaeodactylum tricornutum)是开展微藻生物柴油研究的理想材料。克隆了内源fcp基因簇的多个调控序列(启动子、终止子),构建了包括fcpB启动子-bar基因-fcpA终止子、以及fcpA启动子-多克隆位点(MCS)-fcpA终止子两个表达盒的通用转化载体pfcpA-MCS/fcpB-Bar,其特征是以bar基因作为选择标记,MCS区方便插入一至多个目的基因。新载体可用于三角褐指藻的重组蛋白表达、或油脂代谢相关基因的功能验证和代谢调控研究。     

Sulphation as a metabolic pathway for oestradiol in the sea urchin Strongylocentrotus franciscanus

1. Aerobic incubation of [¹⁴C]oestradiol, in the presence of surviving gut tissue of the sea urchin Strongylocentrotus franciscanus, or a soluble enzyme system prepared therefrom, resulted in rapid formation of a water-soluble metabolite, identified as oestradiol 3-sulphate. 2. No evidence was obtained for the formation of other metabolic derivatives by the urchin-gut enzymes, or for the presence of the sulphating capacity in any other tissues of the organism under the conditions used. 3. The data are consistent with the possibility that oestradiol, previously detected in the gonads, is synthesized therein and excreted in a conjugated, highly water-soluble form via the gut.     

Bacteria associated with a marine planktonic copepod in culture. II. Degradation of fecal pellets produced on a diatom, a nanoflagellate or a dinoflagellate diet

Copepods (A cartia tonsa Dana) were allowed to feed on culturedalgal species simulating the yearly boreal phytoplankton succession,where the spring is characterized by diatoms, the summer bynanoflagellates and the autumn by dinoflagellates. Degradationof freshly produced fecal pellets in suspension was followedover time by volume recording and estimation of carbon loss.The fecal pellets produced on a diatom diet were very denseand showed a significantly slower degradation than those producedon nanoflagellate or dinoflagellate diets. This indicates thatthe algal texture, i.e. the mechanical robustness given by inertstructures, determines the degradation rate of the pellets.The degradation rate was also very dependent on the algal concentrationfor the grazer (565 µg Cl^–1 T₁     

Evaluation of three techniques for the study of harvester ant (Pogonomyrmex spp.) diet

The estimation of an ant's diet is crucial in many ecological studies. Different techniques, which involve different assumptions and field procedures, have been used to estimate the composition of harvester ant diet. In this study, three techniques are compared for the estimation of the diet of Pogonomyrmex rastratus (Mayr), Pogonomyrmex pronotalis (Santschi), and Pogonomyrmex inermis (Forel) in the central Monte desert, Argentina: (1) hand collection of items brought back to the nest by foragers, (2) collection of items with a semiautomated device with pitfall traps, and (3) collection of the discarded material accumulated in middens. The hand collection technique and the collection of middens provided the lowest and the highest number of items, respectively. Midden samples and pitfall traps contained a higher proportion of nonseed items, probably coming from sources other than ants, than hand-collected items. The three techniques provided similar estimations of species richness but a bias against small seeds was detected for P. pronotalis and P. inermis with the hand collection technique, possibly because of the difficulty of collecting small items by hand. The percentage of seed species in the diet obtained with different techniques was positively correlated in the great majority of colonies. Overall, despite their intrinsic differences, the three techniques proved consistent, which constitutes a robustness test for the estimations obtained. In comparative ecological studies, the awareness that results depend on the techniques and their assumptions is particularly important.     

Toxic cyanobacteria Nodularia spumigena in the diet of Baltic mysids: Evidence from molecular diet analysis

A PCR-based method was used to detect toxic cyanobacteria Nodularia spumigena in the diet of Baltic mysids, Mysis mixta and Mysis relicta. The decay in detectability of Nodularia DNA in mysid stomachs and feces following the cyanobacterium consumption was examined in laboratory with special references to (1) marker size (780 bp vs. 200 bp), (2) mysid developmental stage (juveniles vs. subadults), and (3) feeding regime after consuming the cyanobacteria (continuous vs. interrupted feeding). The Nodularia DNA could be reliably detected in mysid stomachs and feces by PCR technique. In the mysid with interrupted feeding, the calculated half-lives of N. spumigena DNA in the mysid stomachs were 1.2 and 6.1 h for 780 and 200 bp fragments, respectively. Continuous feeding, however, facilitated decay in the detectability, most likely due to increased gut evacuation rate. In stomachs of the field-collected mysids, the Nodularia DNA was detected with high frequencies, 60% in M. mixta and 51% in M. relicta. Moreover, it was higher in immature mysids than in adults and correlated with stomach fullness in age-specific manner: in juveniles and subadults, stomachs containing Nodularia were significantly fuller, while in adults, the presence of the cyanobacteria was associated with empty stomachs. This suggests greater habitat overlap for juvenile mysids and N. spumigena and thus higher encounter and consumption rates. These findings contribute to a growing body of evidence that cyanobacteria in the Baltic Sea are important food for grazers.     

Evidence of protocarnivory in triggerplants (Stylidium spp.; Stylidiaceae)

Australian triggerplants (Stylidium spp.; Stylidiaceae) trap small insects using mucilage-secreting glandular hairs held at various points on their inflorescence stems and flower parts. Triggerplants are generally found in habitats also containing genera of plants already accepted as carnivorous, two of which (Drosera, Byblis) use the same basic mechanism as Stylidium to trap their prey. In the herbarium, sheets of triggerplants and of accepted groups of carnivorous plants held similar numbers of trapped insects, and in the field, trapping of small prey per unit of glandular surface area was the same at a given site for triggerplants and for nearby carnivorous plants at three sites in northern Australia. Even more important, protease activity was produced by glandular regions of both triggerplants and Drosera after induction with yeast extract. A panel of negative and positive controls, including use 1) of plants grown in tissue culture, which therefore lack surface microorganisms, and 2) of protease inhibitors, shows that this activity 1) is generated by the glandular regions of the triggerplant itself, not by organisms that might reside on the surface of the plants, and 2) is due to proteases. All of this evidence taken together provides strong evidence of protocarnivory in Stylidium, something not previously suggested in the scientific literature, though the insect trapping has been noted informally. Experiments remain to be done to determine nutrient uptake, so triggerplants may well be fully carnivorous.