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17.4E: Edible Algae - Biology


Edible algae have been used as food for centuries in many coastal regions all over the world.

Learning Objectives

  • Describe the nutritional value of algae

Key Points

  • Algae are a very diverse group of generally simple unicellular or multicellular eukaryotic organisms.
  • Algae are of excellent nutritional value since they contain complete protein, fiber, and sometimes high levels of omega-3 fatty acids, many vitamins and minerals.
  • Some compounds that are used as additives in the food industry are isolated from algae.

Key Terms

  • complete protein: Complete protein (whole protein) is a protein that contains all of the nine essential amino acids.

Algae are a very diverse group of generally simple unicellular or multicellular eukaryotic organisms. Most of them are autotrophic which means that they can harvest carbon dioxide from the atmosphere and convert it to organic matter. They inherited their photosynthetic apparatus from cyanobacteria. Cyanobacteria are sometimes called blue-green algae but they are prokaryotic organisms and are not true algae. Some cyanobacterial species are used as food as well.

Seaweeds are edible algae that have been used for centuries as food in many coastal regions all over the world. They may belong to one of three groups of multicellular algae: red, green or brown. In countries such as China, Japan, Korea and to some extent Iceland, Ireland, Chile and New Zealand algae are part of people’s regular diet. They are usually of marine origin since freshwater algae are often poisonous.

Algae are of excellent nutritional value since they contain complete protein (in contrast to plant food harvested on land), fiber, and sometimes high levels of omega-3 fatty acids. In fact, the omega-3 acids in fish comes from the microalgae consumed at the bottom of the food pyramide and gradually passed up to the fish at the top. Algae are also rich in many vitamins, such as A, C, B1, B2, B3 and B6, as well as minerals, such as iodine, calcium, potassium, magnesium and iron. They are consumed both cooked, dried and raw.

Cultivated microalgae and cyanobacteria such as Spirulina and Chlorella are sold as nutritional supplements. Hydrocolloids such as agar, alginate and carrageenan are isolated from wild and cultivated algae and used as additives in the food industry for their emulsifying and thickening properties. Some of the complex polysaccharides found in algae may be digested by bacteria in the gut since the needed enzymes for digestion are abundantly present in Japanese people but absent in people from North America.


Interferon

Interferons (IFNs, / ˌ ɪ n t ər ˈ f ɪər ɒ n / [1] ) are a group of signaling proteins [2] made and released by host cells in response to the presence of several viruses. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.

IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. [3] Interferons are named for their ability to "interfere" with viral replication [3] by protecting cells from virus infections. IFNs also have various other functions: they activate immune cells, such as natural killer cells and macrophages they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines.

More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all three classes are important for fighting viral infections and for the regulation of the immune system.


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Metabolome profiling of various seaweed species discriminates between brown, red, and green algae

Among seaweed groups, brown algae had characteristically high concentrations of mannitol, and green algae were characterised by fructose. In red algae, metabolite profiles of individual species should be evaluated.

Seaweeds are metabolically different from terrestrial plants. However, general metabolite profiles of the three major seaweed groups, the brown, red, and green algae, and the effect of various extraction methods on metabolite profiling results have not been comprehensively explored. In this study, we evaluated the water-soluble metabolites in four brown, five red, and two green algae species collected from two sites in northern Japan, located in the Sea of Japan and the Pacific Ocean. Freeze-dried seaweed samples were processed by methanol–water extraction with or without chloroform and analysed by capillary electrophoresis- and liquid chromatography-mass spectrometry for metabolite characterisation. The metabolite concentration profiles showed distinctive characteristic depends on species and taxonomic groups, whereas the extraction methods did not have a significant effect. Taxonomic differences between the various seaweed metabolite profiles were well defined using only sugar metabolites but no other major compound types. Mannitol was the main sugar metabolites in brown algae, whereas fructose, sucrose, and glucose were found at high concentrations in green algae. In red algae, individual species had some characteristic metabolites, such as sorbitol in Pyropia pseudolinearis and panose in Dasya sessilis. The metabolite profiles generated in this study will be a resource and provide guidance for nutraceutical research studies because the information about metabolites in seaweeds is still very limited compared to that of terrestrial plants.

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Materials and Methods

Chemicals and Reagents

All chemicals were obtained from from Sigma (St. Louis, MO, USA), unless otherwise stated.

Diatom Materials and Sample Preparation

C. weissflogii ND-8 was isolated from the coastal water of Zhoushan, Zhejian Province in China. It was cultured in Guillard’s f/2 medium prepared from filtered, sterilized natural seawater, with an inoculation ratio of 1:8, under 12 h light condition (light intensity of 75 μmol/m 2 /s) and 12 h dark time in 1 day at 20ଌ�ଌ.

Characterization of C. weissflogii ND-8

The photomicrographs of C. weissflogii ND-8 were taken with an optical microscope (FM 10 Camera Nikon, Tokyo, Japan) and a scanning electron microscope (JSM-6380LV, JEOL, Tokyo, Japan). Molecular identification was performed as previously described (Su and Yang, 2015). Primers ITS5-F (5’-TCACCTACGGAAACCTTGT-3’)/ITS5-R (5’-TTCAGCGGGTAGTCTTGCCTC-3’), and 18S-F (5’-ACCTGGTTGATCCTGCCAGT-3’)/18S-R (5’-TCACCTACGGAAACCTTGT-3’) were used to amplify the ND-8 ITS and 18S fragments, respectively. Their ITS and 18S sequences were compared with those available in the NCBI databases using BLAST. The search results were processed with the MEGA5.2 software (Tamura et al., 2011). The phylogenetic tree was constructed by the neighbor-joining method, 1,000 replications of random search were carried out to assess the reliable level of the tree (Saitou and Nei, 1987).

FX Extraction and Isolation

C. weissflogii ND-8 was grown in Guillard’s f/2 medium at 20ଌ�ଌ for 5 days, followed by centrifugation at 4,000 × g for 15 min. The algae mud was collected, freeze-dried at �ଌ for 2 days. The purification of FX was performed as previously described (Xia et al., 2013). Additionally, to avoid interference of light, all experiments were performed in the dark. The active fractions were pooled by TLC in a solvent system containing petroleum ether/ethyl acetate, 1:1 (v/v). The retention factor (Rf) was calculated as follows:

The active fractions were pooled and concentrated in vacuum. The concentrates were finally dried with a nitrogen blower for subsequent separation and analysis.

HPLC-MS

HPLC-MS analysis was performed on the Thermo HPLC-MS system (Thermo Scientific, Waltham, MA, USA) using the Thermo Hypersil GOLD C18 column (1.9-μm particle size, 2.1 mm × 100 mm) with methanol and water as eluents. The experimental conditions were following: injection volume: 5 μM mobile phase: 0𠄰.2 min, 95% B 0.2𠄳.5 min, 95%𠄲% B 3.5𠄵 min, 2% B 5𠄷.5 min, 2%�% B 7.5� min, 95% B flow rate: 0.3 ml·min 𠄱 . The HPLC eluate was administered to the MS system with a spray voltage of 1.0 kV. The MS peaks were recorded and compared with that of the FX standard.

The isolated target sample (2.0 mg) and standard FX (2.0 mg) were dissolved in 0.5 ml of deuterochloroform (CDCl3) and the 1 H nuclear magnetic resonance (NMR) was measured using the Bruker 400 MHz NMR spectrometer (MA, USA).

Animals and Treatments

Specific pathogen-free C57BL/6 adult mice aged 8� weeks old and weighing 20 ± 1 g were purchased from the Fujian Medical University Animal Facility (Fujian, China). The sample included 126 animals, half male and half female. All animal experiments were conducted in accordance to the Guide for the Care and Use of Laboratory Animals approved by the Fujian Provincial Office for Managing Laboratory Animals and was guided by the Fujian Normal University Animal Care and Use Committee (Approval No. 201800013).

After acclimation for 1 week, 90 mice were randomly divided into nine groups (n = 10/group) with half male and half female, including Sham group (mouse received an intraperitoneal injection of normal saline 1.0 ml/kg), FX-treated (0.1�.0 mg/kg), LPS-treated (20 mg/kg), FX+LPS-treated (0.1�.0 mg/kg and 20 mg/kg, respectively), and urinastatin groups (10 4 U/kg). LPS was obtained from Escherichia coli 0111:B4 cells (Cell Signaling Technology, Beverley, MA, USA). Ulinastatin was used as the positive control. The sham group was injected with the same amount of phosphate-buffered saline (PBS) (0.0067M pH 7.4, HyClone, GE Healthcare Life Sciences, UT, USA). The mice were intraperitoneally (ip) injected with FX 30 min before the ip administration of a lethal dose of LPS (20 mg/kg) or PBS. All mice were fasted for 12 h preoperatively, but were free to drink water. The survival rate of mice was recorded every 6 h for 120 h, and Kaplan–Meier survival curves were generated using the GraphPad Prism 6 software (v.5.01 for Windows GraphPad Software, San Diego, CA, USA) and analyzed by the log-rank test. Based on the above experiments, the other 36 mice were randomly divided into six groups (n = 6/group) with half male and half female. After anesthetization using Pentobarbital sodium salt, mouse blood was drawn via retro-orbital puncture 6-h post-challenge and allowed to clot at 28ଌ for 30 min. The serum was subsequently collected by centrifugation at 2000 ×g for 30 min and stored at �ଌ for further analysis. The mice tissues were collected for further analyses.

Histopathological Examination

Mouse tissues were fixed with paraformaldehyde (4%) in PBS for histological analysis. Tissues were rinsed with water, dehydrated with ethanol, and embedded in paraffin, followed by cryostat sectioning (𢏄 μm) and mounting onto glass slides. Sections were then dewaxed using xylene and ethanol, and stained with general hematoxylin and eosin (H&E) to reveal hemorrhagic necrosis in the tissues. Histological changes were observed under a light microscope (Olympus, Japan) at 100× and 200× magnifications. According to Eriksson et al., hepatic injury score was measured on the H&E-stained sections using grades from 0 to 4 as follows: a score of 0 represented no inflammatory infiltrates 1 represented small inflammatory cells between hepatocytes 2 represented larger foci of >100 inflammatory cells 3 represented >10% of a cross section involved 4 represented >30% of a cross section involved (Eriksson et al., 2003).

Cell Culture

Murine macrophage RAW 264.7 was purchased from the American Type Culture Collection (Manassas, VA, USA) and used as an in vitro model to investigate the anti-inflammatory properties of FX. The cells were cultured in Dulbecco’s modified Eagle medium containing 10% (v/v) fetal bovine serum (Gibco, CA, USA) and 1% (v/v) penicillin/streptomycin (Gibco, CA, USA) at 37ଌ in a humidified incubator with 5% carbon dioxide (CO2).

Cell-Viability Assay

Cell viability was evaluated in RAW 264.7 cells using the Cell Counting Kit-8 (CCK-8 Beyotime Biotechnology, Beijing, China). The absorbance of the sample at 450 nm was measured using a microplate reader (Synergy HT BioTek, Winooski, VT, USA), and the percent of surviving cells in each treated group was plotted.

Real-Time Quantitative PCR

Cytokine mRNA expression and secretion was measured by the real-time quantitative polymerase chain reaction following reverse transcription (RT-qPCR). The total RNA was separated using TRIZOL (Invitrogen, Carlsbad, CA, USA). The RT-qPCR was performed using SYBR Green (Applied Biosystems, Foster City, CA, USA) as previously described (Fan et al., 2018). The primers used for RT-qPCR were following: IL-1β-F (5’-ACAGGCTCCGAGATGAACAA-3’)/IL-1β-R (5’-TGGGAGTAGACAAGGTACAACCC-3’), IL-6-F(5’-TAGTCCTTCCTACCCCAATTTCC-3’)/IL-6-R (5’-CCTCTCGGCAGTGGATAAAG-3’), and TNF-α-F (5’-CATCTTCTCAAAATTCGAGTGACAA-3’)/TNF-α-R (5’-TGGGAGTAGACAAGGTACAACCC-3’).

Measurement of Pro-Inflammatory Cytokine Levels by ELISA

RAW 264.7 cells were pre-treated with FX at the indicated doses for 6 h followed by 3-h LPS (1.0 mg/L) treatment, and grown in 24-well plates (1 × 10 6 cells/well) for 24 h. The supernatants of the cultured RAW 264.7 cells were collected.

The levels of TNF-α, IL-1β, and IL-6 in the supernatants and serum samples (see above) were quantified using the ELISA kits (TNF-α, R&D Systems, Minneapolis, MN, USA, catalog number SMTA00B IL-1β, R&D Systems, Minneapolis, MN, USA, catalog number SMLB00C IL-6, R&D Systems, Minneapolis, MN, USA, catalog number SM6000B) according to manufacturer’s protocols.

Western Blot Analysis

Western blotting was performed as previously described (Chen et al., 2002 Xiao et al., 2019). Antibodies anti-myeloid differentiation primary response gene 88 (MyD88) (D80F5 #4283), anti-phospho-IKKα/β (Ser176/180) (16A6 #2697), anti-IKKα (#2682), Phospho-IKKα/β (Ser176/180) (16A6, #2697), anti-I㮫α (44D4,#4812), anti-Phospho-I㮫α (Ser32/36) (5A5, #9246), anti-NF-㮫 p65 (D14E12, #8242) and anti-Phospho-NF-㮫 p65 (Ser536) (93H1, #3033) were purchased from Cell Signaling Technology.

NF-㮫 Luciferase Activity Assay

Human THP1-Lucia TM NF-㮫 cells were derived from the human THP-1 monocyte cell line by stable integration of an NF-㮫-inducible Lucia TM reporter construct. THP-1 Lucia NF㮫 reporter cells were purchased from InvivoGen (San Diego, CA, USA). THP1-Lucia TM NF-㮫 cells were specifically designed for monitoring the NF-㮫 signal transduction pathway in a physiologically relevant cell line. The THP1-Lucia TM NF-㮫 cells were grown on 96-well plate (1 × 10 5 /well) 18 h in the presence of the different concentrations FX followed by LPS (1.0 mg/L) for stimulation. For the determining of the luciferase activity, the 20 μl aliquots of cell culture media were relocated into the 96-well black plates (Corning, NY, USA) followed by QUANTI-Luc TM assay solution (InvivoGen). Plates were measured immediately for luciferase activity with Victor 2 multiplate reader (PerkinElmer) according to the manufacturer’s instructions.

Immunofluorescent Staining

Cells were grown and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by treatment with membrane penetration solution (0.3% Triton-100) for 10 min at room temperature. The cells were washed with 1 × PBS five times then incubated with anti-NF-㮫 (p65) primary antibodies (1:200 dilution) (Cell Signaling Technology, USA) overnight at 4ଌ, followed by incubation with AlexaFluor 488 goat antirabbit secondary antibody at 37ଌ in the dark for 30 min. Nuclei were counterstained with 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (1:800, Santa Cruz) in PBS for 2 min. Negative controls were prepared by omitting primary antibodies. After washing with PBS three times, samples were mounted in mounting medium (M1289, Sigma-Aldrich), observed under a Zeiss fluorescence microscope (Carl Zeiss, Oberkochen, Germany), and image analyses were performed using Zeiss LSM 510 software.

Statistical Analyses

The data are expressed as mean ± standard deviation (SD). Statistical significance was determined by the one-way ANOVA and Tukey’s test for post hoc multiple comparison by 5 software. The P value < 0.05 was considered statistically significant.


17.4E: Edible Algae - Biology

The application of LC-NMR/MS for the direct identification of carbohydrates in beer has been studied. Carbohydrates are major beer components, and their structural characterization by NMR alone is seriously hindered by strong spectroscopic overlap. Direct analysis of beer by LC-NMR/MS enables the rapid (1−2 h) identification of dextrins with degree of polymerization (DP) of up to nine monomers, with degassing being the only sample treatment required. Although the presence of α(1→6) branching points is easily indicated by NMR for each subfraction separated by LC, difficulties arise for the unambiguous assignment of linear or branched forms of high DP dextrins. The two beer samples investigated in this work were found to have significantly different oligosaccharide compositions, reflecting the different production conditions employed. The use of hyphenated NMR for the rapid characterization of the carbohydrate composition of beers may be the basis of a useful tool for the quality control of beer.

Keywords: Beer NMR LC-NMR/MS carbohydrate dextrins composition

Extraction of Chili, Black Pepper, and Ginger with Near-Critical CO2, Propane, and Dimethyl Ether: Analysis of the Extracts by Quantitative Nuclear Magnetic Resonance
  • Owen J. Catchpole ,
  • John B. Grey ,
  • Nigel B. Perry ,
  • Elaine J. Burgess ,
  • Wayne A. Redmond , and
  • Noel G. Porter

Ginger, black pepper, and chili powder were extracted using near-critical carbon dioxide, propane, and dimethyl ether on a laboratory scale to determine the overall yield and extraction efficiency for selected pungent components. The temperature dependency of extraction yield and efficiency was also determined for black pepper and chili using propane and dimethyl ether. The pungency of the extracts was determined by using an NMR technique developed for this work. The volatiles contents of ginger and black pepper extracts were also determined. Extraction of all spice types was carried out with acetone to compare overall yields. Subcritical dimethyl ether was as effective at extracting the pungent principles from the spices as supercritical carbon dioxide, although a substantial amount of water was also extracted. Subcritical propane was the least effective solvent. All solvents quantitatively extracted the gingerols from ginger. The yields of capsaicins obtained by supercritical CO2 and dimethyl ether were similar and approximately double that extracted by propane. The yield of piperines obtained by propane extraction of black pepper was low at ∼10% of that achieved with dimethyl ether and CO2, but improved with increasing extraction temperature.

Keywords: Spices ginger black pepper chili extraction near-critical pungent gingerol piperine capsaicin quantitative NMR

Simultaneous Liquid Chromatographic Determination of Creatinine and Pseudouridine in Bovine Urine and the Effect of Sample pH on the Analysis

A rapid, reliable method for the simultaneous determination of creatinine and pseudouridine is described. Both analytes were detected at an optimum wavelength of detection (262 nm), considering the relative levels present in bovine urine. Cimetidine was used as the internal standard and detected at its maximum wavelength of absorption (220 nm) on a separate channel. All three compounds were eluted within 15 min, using a 10 mmol/L phosphate buffer (pH 6.8)-methanol gradient on a C18 column. Creatinine data were found to be significantly dependent upon the pH of the sample. Recoveries of both analytes were above 96%. Lowest detectable levels of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, respectively. The use of internal standard resulted in a method with high precision (standard deviation of 1.42 mmol/L and 0.027 mmol/L for creatinine and pseudouridine), yet one that was simple and rapid.

Keywords: Creatinine pseudouridine HPLC diode-array internal standard cimetidine

Differentiation of Natural and Synthetic Phenylalanine and Tyrosine through Natural Abundance 2 H Nuclear Magnetic Resonance
  • Elisabetta Brenna ,
  • Giovanni Fronza ,
  • Claudio Fuganti , and
  • Matteo Pinciroli

The natural abundance deuterium NMR characterization of samples of the amino acids tyrosine (1) and phenylalanine (2), examined as the acetylated methyl esters 4 and 6, has been performed with the aim to identify by these means the contribution in animals of the hydroxylation of the diet l-phenylalanine (2) to the formation of l-tyrosine (1), a feature previously revealed on the same samples through the determination of the phenolic δ18O values. The study, which includes also the NMR examination of benzoic acid (5) from 2 and of tyrosol (7) from 1, substantially fails in providing the required information because the mode of deuterium labeling of tyrosine samples of different origins is quite similar but indicates a dramatic difference in the deuterium labeling pattern of the two amino acids 1 and 2. The most relevant variation is with regard to the deuterium enrichments at the CH2 and CH positions, which are inverted in the two amino acids of natural derivation. Moreover, whereas the diastereotopic benzylic hydrogen atoms of l-tyrosine (1) appear to be equally deuterium enriched, in l-phenylalanine (2) the (D/H)3R > (D/H)3S. Similarly, benzoic acid (5) shows separate signals for the aromatic deuterium nuclei, which are quite indicative of the natural or synthetic derivation. The mode of deuterium labeling of the side chain of 1 and 2 is tentatively correlated to the different origins of the two amino acids, natural from animal sources for l-tyrosine and biotechnological probably from genetically modified microorganisms for l-phenylalanine.

Keywords: Tyrosine phenylalanine tyrosol animal vegetal origin aspartame natural abundance deuterium NMR

Identification of Volatile Compounds in Soybean at Various Developmental Stages Using Solid Phase Microextraction
  • Stephen M. Boué ,
  • Betty Y. Shih ,
  • Carol H. Carter-Wientjes , and
  • Thomas E. Cleveland

Soybean (Glycine max) seed volatiles were analyzed using a solid phase microextraction (SPME) method combined with gas chromatography−mass spectrometry (GC-MS). Thirty volatile compounds already reported for soybean were recovered, and an additional 19 compounds not previously reported were identified or tentatively identified. The SPME method was utilized to compare the volatile profile of soybean seed at three distinct stages of development. Most of the newly reported compounds in soybean seed were aldehydes and ketones. During early periods of development at maturity stage R6, several volatiles were present at relatively high concentrations, including 3-hexanone, (E)-2-hexenal, 1-hexanol, and 3-octanone. At maturity stage R7 and R8, decreased amounts of 3-hexanone, (E)-2-hexenal, 1-hexanol, and 3-octanone were observed. At maturity stage R8 hexanal, (E)-2-heptenal, (E)-2-octenal, ethanol, 1-hexanol, and 1-octen-3-ol were detected at relatively high concentrations. SPME offers the ability to differentiate between the three soybean developmental stages that yield both fundamental and practical information.

Keywords: Soybean volatiles maturity gas chromatography mass spectrometry solid phase microextraction SPME

Isotopic Labeling and LC-APCI-MS Quantification for Investigating Absorption of Carotenoids and Phylloquinone from Kale (Brassica oleracea)
  • Anne C. Kurilich ,
  • Steven J. Britz ,
  • Beverly A. Clevidence , and
  • Janet A. Novotny

The ability to study bioavailability of nutrients from foods is an important step in determining the health impact of those nutrients. This work describes a method for studying the bioavailability of nutrients from kale (Brassica oleracea var. Acephala) by labeling the nutrients with carbon-13, feeding the kale to an adult volunteer, and analyzing plasma samples for labeled nutrients. Results showed that conditions for producing atmospheric intrinsically labeled kale had no detrimental effect on plant growth. Lutein, β-carotene, retinol, and phylloquinone were analyzed using liquid chromatography−atmospheric pressure chemical ionization mass spectrometry. Analysis of plasma samples showed that labeled lutein peaked in plasma at 11 h (0.23 μM), β-carotene peaked at 8 (0.058 μM) and 24 h (0.062 μM), retinol peaked at 24 h (0.10 μM), and phylloquinone peaked at 7 h (3.0 nM). This method of labeling kale with 13C was successful for producing clearly defined kinetic curves for 13C-lutein,13C-β-carotene, 13C-retinol, and 13C-phylloquinone.

Keywords: Carotenoid β-carotene lutein retinol vitamin A phylloquinone vitamin K isotope label mass spectrometry LC-MS kale Brassica oleracea

BIOACTIVE CONSTITUENTS
Ovicidal and Adulticidal Effects of Eugenia caryophyllata Bud and Leaf Oil Compounds on Pediculus capitis
  • Young-Cheol Yang ,
  • Si-Hyeock Lee ,
  • Won-Ja Lee ,
  • Don-Ha Choi , and
  • Young-Joon Ahn

The toxicity of Eugenia caryophyllata bud and leaf oil-derived compounds (acetyleugenol, β-caryophyllene, eugenol, α-humulene, and methyl salicylate) and congeners of eugenol (isoeugenol and methyleugenol) against eggs and females of Pediculus capitis was examined using direct contact application and fumigation methods and compared with those of the widely used δ-phenothrin and pyrethrum. In a filter paper diffusion bioassay with female P. capitis, the pediculicidal activity of the Eugenia bud and leaf oils was comparable to those of δ-phenothrin and pyrethrum on the basis of LT50 values at 0.25 mg/cm2. At 0.25 mg/cm2, the compound most toxic to female P. capitis was eugenol followed by methyl salicylate. Acetyleugenol, β-caryophyllene, α-humulene, isoeugenol, and methyleugenol were not effective. Eugenol at 0.25 mg/cm2 was as potent as δ-phenothrin and pyrethrum but was slightly less effective than the pyrethroids at 0.125 mg/cm2. Against P. capitis eggs, methyl salicylate and eugenol were highly effective at 0.25 and 1.0 mg/cm2, respectively, whereas little or no activity at 5 mg/cm2 was observed with the other test compounds as well as with δ-phenothrin and pyrethrum. In fumigation tests with female P. capitis at 0.25 mg/cm2, eugenol and methyl salicylate were more effective in closed cups than in open ones, indicating that the effect of the compounds was largely due to action in the vapor phase. Neither δ-phenothrin nor pyrethrum exhibited fumigant toxicity. The Eugenia bud and leaf essential oils, particularly eugenol and methyl salicylate, merit further study as potential P. capitis control agents or lead compounds.

Keywords: Natural insecticide pediculicide ovicide fumigant Pediculus capitis Eugenia caryophyllata essential oil GC-MS eugenol methyl salicylate mode of action

Anthocyanin and Proanthocyanidin Content in Selected White and Red Wines. Oxygen Radical Absorbance Capacity Comparison with Nontraditional Wines Obtained from Highbush Blueberry

Antioxidant capacity, as measured by oxygen radical absorbance capacity (ORACPE), total phenolic, total and individual anthocyanins, and proanthocyanidin fraction contents were evaluated in red and white wines from grapes. A comparison in terms of antioxidant capacity is made with nontraditional wines made from highbush blueberry. Blueberries are among fruits that are best recognized for their potential health benefits. In red wines, total oligomeric proanthocyanidin content, including catechins, was substantially higher (177.18 ± 96.06 mg/L) than that in white wines (8.75 ± 4.53 mg/L). A relative high correlation in red wines was found between ORACPE values and malvidin compounds (r = 0.75, P < 0.10), and proanthocyanidins (r = 0.87, P < 0.05). In white wines, a significant correlation was found between the trimeric proanthocyanidin fraction and peroxyl radical scavenging values (r = 0.86, P < 0.10). A moderate drink (1 drink per day, about 140 mL) of red wine, or white wine, or wine made from highbush blueberry corresponds to an intake of 2.04 ± 0.81 mmol of TE, 0.47 ± 0.15 mmol of TE, and 2.42 ± 0.88 mmol of TE of ORACPE/day, respectively.

Keywords: Wine grape blueberry anthocyanins proanthocyanidins phenolics ORAC

Isolation and Antihypertensive Effect of Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Spinach Rubisco
  • Yanjun Yang ,
  • Ewa D. Marczak ,
  • Megumi Yokoo ,
  • Hachiro Usui , and
  • Masaaki Yoshikawa

Four new inhibitory peptides for angiotensin I-converting enzyme (ACE), that is, MRWRD, MRW, LRIPVA, and IAYKPAG, were isolated from the pepsin−pancreatin digest of spinach Rubisco with the use of HPLC. IC50 values of individual peptides were 2.1, 0.6, 0.38, and 4.2 μM, respectively. MRW and MRWRD had an antihypertensive effect after oral administration to spontaneously hypertensive rats. Maximal reduction occurred 2 h after oral administration of MRW, whereas MRWRD showed maximal decrease 4 h after oral administration at doses of 20 and 30 mg/kg, respectively. IAYKPAG also exerted antihypertensive activity after oral administration at the dose of 100 mg/kg, giving a maximum decrease 4 h after oral administration. IAYKP, IAY, and KP, the fragment peptides of IAYKPAG, also exerted antihypertensive activity. LRPVIA did not show any antihypertensive effect at a dose of 100 mg/kg despite its potent ACE-inhibitory activity.

Keywords: Rubisco spinach ACE-inhibitory peptides antihypertensive effect spontaneously hypertensive rats (SHR)

Chemical Characterization and Biological Effects of Sicilian Opuntia ficus indica (L.) Mill. Fruit Juice: Antioxidant and Antiulcerogenic Activity
  • Enza Maria Galati ,
  • Maria Rita Mondello ,
  • Daniele Giuffrida ,
  • Giacomo Dugo ,
  • Natalizia Miceli ,
  • Simona Pergolizzi , and
  • Maria Fernanda Taviano

The juice of whole fruits of Sicilian cultivars of prickly pear (Opuntia ficus indica (L.) Mill.) was investigated, and the contents of ascorbic acid, total polyphenols, and flavonoids were determined. In the juice, ferulic acid was the chief derivative of hydroxycinnamic acid and the mean concentration of total phenolic compounds was 746 μg/mL. The flavonoid fraction, analyzed by high-performance liquid chromatography−diode array detection, consisted of rutin and isorhamnetin derivatives. The juice showed antioxidant activity in the DPPH• test, probably due to the phenolic compounds that are effective radical scavengers. The preventive administration of the juice inhibited the ulcerogenic activity of ethanol in rat. Light microscopy observations showed an increase in mucus production and the restoration of the normal mucosal architecture. The juice is nutritionally interesting, and its dietary intake could provide protection against oxidative damage.

Keywords: Opuntia ficus indica fruit juice antioxidant flavonols antiulcer activity mucosa structural changes

Enhancing Volatile Phenol Concentrations in Wine by Expressing Various Phenolic Acid Decarboxylase Genes in Saccharomyces cerevisiae
  • Annél Smit ,
  • Ricardo R. Cordero Otero ,
  • Marius G. Lambrechts ,
  • Isak S. Pretorius , and
  • Pierre van Rensburg

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1P) and terminator (PGK1T) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Σ1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Σ1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.

Keywords: Phenolic acid decarboxylation volatile phenols wine yeast wine aroma

Molluscicidal Saponins from Sapindus mukorossi, Inhibitory Agents of Golden Apple Snails, Pomacea canaliculata
  • Hui-Chi Huang ,
  • Sin-Chung Liao ,
  • Fang-Rong Chang ,
  • Yao-Haur Kuo , and
  • Yang-Chang Wu

Extracts of soapnut, Sapindus mukorossi Gaertn. (Sapindaceae) showed molluscicidal effects against the golden apple snail, Pomacea canaliculata Lamarck. (Ampullariidae) with LC50 values of 85, 22, and 17 ppm after treating 24, 48, and 72 h, respectively. Bioassay-directed fractionation of S. mukorossi resulted in the isolation of one new hederagenin-based acetylated saponin, hederagenin 3-O-(2,4-O-di-acetyl-α-l-arabinopyranoside)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (1), along with six known hederagenin saponins, hederagenin 3-O-(3,4-O-di-acetyl-α-l-arabinopyranoside)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (2), hederagenin 3-O-(3-O-acetyl-β-d-xylopyranosyl)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (3), hederagenin 3-O-(4-O-acetyl-β-d-xylopyranosyl)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (4), hederagenin 3-O-(3,4-O-di-acetyl-β-d-xylopyranosyl)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (5), hederagenin 3-O-β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (6), and hederagenin 3-O-α-l-arabinopyranoside (7). The bioassay data revealed that 1−7 were molluscicidal, causing 70−100% mortality at 10 ppm against the golden apple snail.

Keywords: Sapindus mukorossi Sapindaceae molluscicidal effects Pomacea canaliculata hederagenin saponins


5. Related Species of Malus domestica

As mentioned earlier, the number of species in the genus Malus varies widely, with different taxonomic treatments recognizing anywhere from 8 to 78 primary species (see Section 2.1). Many of the crabapple species can be difficult to differentiate due to the lack of distinguishing characters (Dickson et al. 1991).

Table 2: Provincial distribution of Malus species present in Canada outside of cultivation (from: Brouillet et al. 2010+ CFIA and NRCan/CFS 2011+ Kartesz 1999 Scoggan 1979).
Descriptive text:

The purpose of the table is to illustrate the provincial distribution of Malus species present in Canada outside of cultivation. The table describes whether the species is native to Canada or introduced and the species distribution by province.

Distribution collated from reports of synonyms Malus pumila Mill. and Malus sylvestris (L. ) Mill. in the literature.

5.1 Inter-species/genus hybridization

Table 3: Reports of experimental interspecific hybrid crosses reported for Malus species present in Canada.
Descriptive text:

The purpose of the table is to highlight reports of experimental interspecific hybrid crosses reported for the Malus species present in Canada. It describes the cross, the number of pollinations and matured fruits, as well as provides references for each cross.

5.2 Potential for introgression of genetic information from Malus domestica into relatives

5.3 Summary of the ecology of relatives of Malus domestica


Material and methods

In this study, a total of 34 PEPC protein sequences of 14 different families in C4 and CAM plants were collected from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov).

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 10) database (http://string-db.org/) was used to foresee the interacting proteins (Szklarczyk et al. 2014 ). The database contains information from numerous sources, including experimental repositories, computational prediction methods and public text collections.

Various online web services and software were used for analyses of PEPC proteins in C4 and CAM plants. Comparative and bioinformatic analyses were carried out online at the website ExPASy (http://expasy.org/tools). Functional domains in PEPC proteins were identified by ProDom server (http://prodom.prabi.fr/prodom/current/html/form.php) (Altschul et al. 1997 ). Physico-chemical parameters of PEPC proteins were analyzed by ProtParam (http://web.expasy.org/protparam) (Gasteiger et al. 2005 ) and the secondary structure prediction was analyzed by SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) (Geourjon and Deleage 1995 ).

Prediction of mitochondrial and plastid targeting sequences was accomplished by Predotar 1.03 server (https://urgi.versailles.inra.fr/predotar/predotar.html) while prediction of signal peptide cleavage sites was achieved by SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP) (Petersen et al. 2011 ). Identification of subcellular locations and transmembrane helices in proteins was performed by TargetP 1.1 and (www.cbs.dtu.dk/services/TargetP/) (Emanuelsson et al. 2000 ) and TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) (Moller et al. 2001 ) servers, respectively.

The tertiary structure prediction analysis of PEPC proteins was performed by the Phyre2 server using profile–profile matching and secondary structure (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Kelley and Sternberg 2009 ). Chimera 1.10.1 was used for 3D structure visualization of Zea mays as the model of plants (https://www.cgl.ucsf.edu/chimera/). Backbone similarities and differences of obtained models were estimated by TM-score server (http://zhanglab.ccmb.med.umich.edu/TM-score/) (Zhang and Skolnick 2004 Xu and Zhang 2010 ). Additionally, the SuperPose web server was used to find the most and the least similarities of PEPC sequences in C4 and CAM plants (http://wishart.biology.ualberta.ca/SuperPose/) (Maiti et al. 2004 ). Finally, stereochemical quality and accuracy of the model was evaluated with PROCHECK 3.5 (http://www.ebi.ac.uk/thornton-srv/software/PROCHECK) by Ramachandran plot analysis (Laskowski et al. 1993 ). Z-score was calculated using interactive ProSA-web service (https://prosa.services.came.sbg.ac.at/prosa.php) for the recognition of errors in three-dimensional structure which indicated model quality and total energy deviation of the structure with respect to energy distribution derived from random conformations (Wiederstein and Sippl 2007 ).

The motifs of protein sequences were discovered using the program of Multiple Em for Motif Elicitation (MEME version 4.10.2) (Bailey et al. 2009 ) and Motif Alignment and Search Tool (MAST version 4.9.1) (Bailey and Gribskov 1998 ) at the website http://meme.nbcr.net/meme. The parameters of MEME analyses were applied as follows: distribution of motif occurrences, zero or one per sequence number of different motifs, 10 minimum motif width, six and maximum motif width, 50.

The multiple sequence alignment of PEPC proteins was performed with ClustalW algorithm implemented in Molecular Evolutionary Genetic Analysis (MEGA 6.06) (http://www.megasoftware.net) (Tamura et al. 2013 ) with default parameters. The phylogenetic tree was constructed using the neighbor-joining (NJ) method and the bootstrap test carried out with 1000 replicates.


DISCUSSION

This study expanded our understanding of the anatomy and function of the EO in two species of planktivorous fishes of special importance, H. molitrix and H. nobilis, by demonstrating that the EO is an important chemosensory organ in these species. We suggest that it uses this sense to accumulate tiny food particles. In addition, this study provides the first detailed description of the morphology of the gill rakers and EO in these important species and shows that their extremely fine gill rakers and EO canals are closely associated and capable of functioning as an integrated unit. While differences in gill raker morphology and spacing were noted between species, their EOs seem much the same (if not identical). We also present histological evidence suggesting that the EO functions as a sophisticated pharyngeal chemosensory organ with both taste buds and SCCs. Our electrophysiological recordings, the first reported from an EO, demonstrate that the EO detects food-related chemicals including l -amino acids and likely other yet unknown chemicals. Together, these data suggest that the EO via pump filtering identifies and packages planktonic food that bigheaded carp have become specialized to consume. This ability might explain the extreme efficiency with which these carp species feed and thus their invasiveness in eutrophic waters.

Histological staining with nuclear red/light green/orange of 14 μm cryosections of the EO. Cartilage and bone were stained green, the brain, nerves and nerve fiber bundles were stained orange, and epithelia including taste buds were stained purple. These stainings were done for a general overview on sections adjacent to the sections used for immunohistochemistry. Images A, B and C show H. molitrix images D, E, F and G show H. nobilis. A, B and C depict cross-sections through the epibranchial organ and its tubes (T). Image B shows the supporting cartilaginous structures (*) and the ridges at the outside of the epibranchial organ (arrowheads). (D) Horizontal section: the tubes are lined with epithelium that contains taste buds (arrows in C). In some areas modified gill rakers face the areas with taste buds. (E) Higher magnification of the modified gill rakers. (F) Some areas of the tubes contain small flaps as shown in Fig. 4C. (G) Higher magnification of the small flaps, which are lined with abundant mucus cells (arrows).

Histological staining with nuclear red/light green/orange of 14 μm cryosections of the EO. Cartilage and bone were stained green, the brain, nerves and nerve fiber bundles were stained orange, and epithelia including taste buds were stained purple. These stainings were done for a general overview on sections adjacent to the sections used for immunohistochemistry. Images A, B and C show H. molitrix images D, E, F and G show H. nobilis. A, B and C depict cross-sections through the epibranchial organ and its tubes (T). Image B shows the supporting cartilaginous structures (*) and the ridges at the outside of the epibranchial organ (arrowheads). (D) Horizontal section: the tubes are lined with epithelium that contains taste buds (arrows in C). In some areas modified gill rakers face the areas with taste buds. (E) Higher magnification of the modified gill rakers. (F) Some areas of the tubes contain small flaps as shown in Fig. 4C. (G) Higher magnification of the small flaps, which are lined with abundant mucus cells (arrows).

The primary finding of this study is that the EO of bigheaded carps functions as a chemoreceptive organ. It has large numbers of taste buds and SCCs and is innervated by vagal nerve fibers that respond to relevant chemical stimuli. Specifically, we found large numbers of both taste buds and SCCs both within and outside the EO in both species. The taste buds stained with calretinin, typical of taste buds described in other fish (Reutter et al., 1974). In contrast, we found few taste buds and no SCCs either on the lips or within the buccal cavity of either species, consistent with our inability to record neural activity to chemical stimuli applied to these areas. Together, these data strongly suggest that the EO is the primary taste organ in these species. Although rather small, taste buds of the bigheaded carps resembled those of other fish species (Reutter et al., 1974). For comparison, taste buds in catfish of sizes comparable to the size of our specimens are about 50–80 μm high (Kirino et al., 2013) and in other fish species reach heights even up to 80 μm and widths 40–60 μm (Hansen and Reutter, 2004). The taste buds in the bigheaded carps were only 22–25 μm high and 10–18 μm wide. That these taste buds responded to AAs is typical of those on other organs in other fishes (Sorensen and Caprio, 1998) (see below). Our experiments also described the presence of SCCs on the EO as well as on the internal flaps within the EO and the gill rakers. SCCs have been observed on gill rakers in other teleosts (Hansen, 2005) as well as other organs of other vertebrates (Finger et al., 2003), but their function is not well understood. SCC morphology varies with respect to the apical endings of the cells and may vary even in the same fish species (Kotrschal et al., 1997), as seen here in the bigheaded carps. Only in the sea robin, Prinotus carolinus (Silver and Finger, 1984), is there direct evidence for the types of chemical stimuli detected by SCCs in fish, and l -amino acids have been implicated. It is possible, but unknown, whether our electrophysiological recordings included responses from SCCs.

In addition to presenting clear histological results that the EO serves as a specialized pharyngeal taste organ, we present electrophysiological evidence that it is responsive to the chemical stimuli found in their planktonic foods. It is notable that the detection threshold of the EO was about 1% that of stock concentration because this concentration would be relevant within the buccal cavity, which lacks other chemosensory structures: the EO appears to be the primary taste organ in these species. The mixture of l -amino acids found in their algal food was only partly responsible for the responsiveness, strongly suggesting that additional unidentified stimuli exist. This is notable because it is commonly thought that l -amino acids are the primary feeding cues in fishes, although most work has focused on carnivores (Sorensen and Caprio, 1998) likely some not yet tested chemostimulatory metabolites are present in their specialized planktonic diet which includes cyanobacteria. Although future studies should examine the physiological function of the EO in greater detail, we believe our work establishes the EO as a new type of internal, pharyngeal taste organ.

Histology of the epibranchial organ. Sections adjacent to those of Fig. 5 were treated with antibodies against calretinin (red), a marker for taste buds and solitary chemosensory cells, and acetylated tubulin (green), a marker for nerve fibers. Images A, B, D and E show H. molitrix, images C, F, G and H show H. nobilis. (A) As seen in Fig. 5, parts of the tubes are lined with epithelium containing taste buds whereas taste buds in other areas are scarce. (B) The epithelium outside the EO has ridges that contain taste buds (cf. Fig. 5B). (C) Taste buds innervated by small tubulin-positive nerve fibers lie opposite modified gill rakers (*), which usually do not have taste buds. (D) The modified gill rakers (*) contain few solitary chemosensory cells (arrow). (E) Higher magnification of the modified gill rakers. The red dots depict a few calretinin-positive solitary chemosensory cells. (F) The ridges on the outside of the EO have small protrusions that contain several taste buds. (G) Higher magnification of a taste bud (red) contacted by small nerve fibers (green). (H) Higher magnification of a solitary chemosensory cell also contacted by small nerve fibers.

Histology of the epibranchial organ. Sections adjacent to those of Fig. 5 were treated with antibodies against calretinin (red), a marker for taste buds and solitary chemosensory cells, and acetylated tubulin (green), a marker for nerve fibers. Images A, B, D and E show H. molitrix, images C, F, G and H show H. nobilis. (A) As seen in Fig. 5, parts of the tubes are lined with epithelium containing taste buds whereas taste buds in other areas are scarce. (B) The epithelium outside the EO has ridges that contain taste buds (cf. Fig. 5B). (C) Taste buds innervated by small tubulin-positive nerve fibers lie opposite modified gill rakers (*), which usually do not have taste buds. (D) The modified gill rakers (*) contain few solitary chemosensory cells (arrow). (E) Higher magnification of the modified gill rakers. The red dots depict a few calretinin-positive solitary chemosensory cells. (F) The ridges on the outside of the EO have small protrusions that contain several taste buds. (G) Higher magnification of a taste bud (red) contacted by small nerve fibers (green). (H) Higher magnification of a solitary chemosensory cell also contacted by small nerve fibers.

Our study extends our understanding of the gross morphology of the EO in bigheaded carps while elaborating on its remarkable anatomical specialization. In particular, we confirmed Boulenger's (Boulenger, 1901) century-old observation that the EO in bigheaded carp contains four blind tubes and demonstrate that the gill rakers of these species directly continue into these tubes through a series of specialized protrusions, probably allowing it to serve as an integrated feeding system. These protrusions, which have not been noted before, may keep larger undesirable particles from entering the EO. As long suspected, but not previously demonstrated, our histological results show that the EO contains large numbers of mucus cells and food boli inside the EO, suggesting that it does indeed aggregate food particles (Wilamovski, 1972). No other specialized secretory cell types were found in the EO, adding no support to a previous conjecture that the organ may also have a digestive function (Bertmar et al., 1969). While confirming an earlier report that the EO is muscular (Wilamovski, 1972 Bauchot et al., 1993), we found new evidence that the EO is reinforced with cartilage, which probably facilitates its ability to forcefully intake and expel water and food particles. Additionally, we illustrated gill raker morphology in both species in a detail not previously shown (Boulenger, 1901 Fang, 1928). Their fine structure is consistent with the likelihood that the gill rakers function with the EO to direct food for aggregation at the entrance of the alimentary canal via cross-flow filtration (Sandersen et al., 2001).

Finally, our study adds new insight into the function of the EO. We show that the EO contains numerous mucus and chemosensory cells, its canals are continuous with the gill rakers, and it contracts strongly when exposed to chemical stimuli. These findings directly support conjecture by Wilamovski (Wilamovski, 1972) that the EO in the bigheaded carps aggregate food from the gill rakers by secreting mucous and pumping and expelling it as boli, to the floor of the pharynx near the tiny alimentary canal for consumption (see Fig. 8 for schematic detail). From this study it now appears that the EO detects the presence of accumulating, desirable food particles in its canals using food chemicals detected by its taste buds and SCCs. Given the huge mass of fine particles that frequently exist in eutrophic (and dimly lit) waters, many of which would not be expected to be edible, but which would tend to accumulate in the gill rakers, the presence of chemosensory cells to discern food consumption would be highly adaptive. The fact that EO detects compounds other than AAs is intriguing given that many phytoplankton species (i.e. cyanobacteria) contain toxins (Beveridge et al., 1993 Leflaive and Ten-Hage, 2007), which might also be detected as part of a possible role of the EO in food selection. Whether mucus production in the EO might be directly stimulated by appropriate food chemicals is unknown. The precise connection between the presence of food particles and their chemicals, and EO pumping will be critical to unravel. Analogies may exist between the EO and the palatal organ, a specialized internal food recognition and sorting system in Eurasian carps including the goldfish, Carassius auratus (Finger, 2008), and common carp (Sibbing, 1982). It is interesting that taste buds and SCCs both occur on the EO, but any functional consequences of this association are unknown at present.

Electrophysiological responses of a branch of the vagus nerve which innervates the epibranchial organ of bighead carp to chemical feeding stimuli. (A) Integrated gustatory electrophysiological responses from a branch of the vagus nerve in bighead carp to increasing concentrations of the filtrate of the algal food (AF) and the l -amino acids this food contains (AA). Mean responses (± s.e.m.) are expressed relative to those elicited by the AA mixture (STD). Data represent three preparations. (B) Representative integrated traces from one of the carp whose data are represented in panel A. Responses are shown to 0.1, 1.0, 10.0, 33.3 and 100% AF (Control: well water control STD: AA mixture at full strength). The responses elicited to 100% AA and AF differed (P<0.10, paired t-test N=3).

Electrophysiological responses of a branch of the vagus nerve which innervates the epibranchial organ of bighead carp to chemical feeding stimuli. (A) Integrated gustatory electrophysiological responses from a branch of the vagus nerve in bighead carp to increasing concentrations of the filtrate of the algal food (AF) and the l -amino acids this food contains (AA). Mean responses (± s.e.m.) are expressed relative to those elicited by the AA mixture (STD). Data represent three preparations. (B) Representative integrated traces from one of the carp whose data are represented in panel A. Responses are shown to 0.1, 1.0, 10.0, 33.3 and 100% AF (Control: well water control STD: AA mixture at full strength). The responses elicited to 100% AA and AF differed (P<0.10, paired t-test N=3).

In conclusion, this study demonstrates new aspects of the function of the EO in fishes, and in bigheaded carps in particular. Our results show that the EO is chemosensitive, and suggest that it plays a role in ingestion and food selection. It is possible that the chemosensitivity of the EO function might be exploited using flavored nanoparticles that are now being considered as means to selectively deliver toxins to these species for control (Hinterthuer, 2012). Further studies will need to determine the full range of chemical classes detected by the EO and its precise role in food ingestion in these species and other species that possess this intriguing organ. How this system might work together with the sense of smell (which is seemingly well developed) to locate, select and ingest novel planktonic food will also be interesting to determine.


Advanced BioFuels USA

Teachers will find many useful links and information on the Education Resources page and on the Grants page. In addition, Advanced Biofuels USA has prepared PowerPoint presentations available in the Biofuels Basics section on the PowerPoint Presentations page.

This page provides links and information about a sample of programs and activities in schools stories about teachers and students who “learned by doing” and news reports about educational activities.

For more examples, click on categories such as Education and Teacher Resources along the right margin of the web site. And, subscribe to our free monthly newsletter.

Network, Share, Collaborate

Advanced Biofuels USA is gathering contact information for educators around the world who are working on curriculum-based educational materials to teach about advanced biofuels. If you or someone you know is working on such materials–or wants to, please provide us with contact information and the reason you/they want to be a part of this network. Let us know what sorts of materials or services this collaborative network might provide that would be useful for developing quality, effective, up-to-date educational materials. Email us at [email protected] and put Educational Network in the subject line.

The Bioenergy and Bioproducts Education Programs (BBEP) (formerly Northeast Bioenergy & Bioproducts (NBB) Programs)

Based at Cornell University, Bioenergy & Bioproducts Education Programs provide professional development and hands-on teaching tools for educators (grades 6 – 16 in service and pre-service teachers and extension educators) who want to learn and teach about the Bioenergy and Bioproducts systems currently in use and under development in the United States. Through the collaborative efforts and expertise of six institutions of research and higher learning, this program aims to inspire today’s students to pursue careers in math and science by aligning concern for the natural environment with the emerging bioenergy and bioproducts industries. READ MORE

Smithsonian Science Education Academies for Teachers

SSEAT Academies provide teachers with an opportunity to take part in a week-long professional development course behind-the-scenes at Smithsonian museums and other world class research facilities throughout the greater Washington, DC area.

Since 2005, the Smithsonian Science Education Center (SSEC) has held summer academies, Smithsonian Science Education Academies for Teachers (SSEATs). The SSEAT Academies provide teachers with an opportunity to take part in a week-long professional development course behind-the-scenes at Smithsonian museums and other world class research facilities throughout the greater Washington, DC area. The academies help to bridge the gap between the formal science education programs of the SSEC and the informal science education that exists throughout the Smithsonian, and combine training in science pedagogy with content presented by scientists and researchers who are experts in their fields.

Recent topics of the SSEATs include Biodiversity, Energy’s Innovations and Implications, Earth’s History and Global Change, and Space Science. Each SSEAT academy engages 20-30 teachers from grades K–12 over a week. Each day, participants engage in carefully selected science experiences directly related to concepts addressed in the content and pedagogical training, and guided by scientists, curators, and educators from a variety of facilities including the Smithsonian. The goals and design of the academy align with the Smithsonian’s goals and the Smithsonian’s Strategic Plan for Science. The Smithsonian Institution strongly supports the initiative, which it sees as complementing its education outreach work of promoting and providing a framework for the participation of their staff in the professional development of teachers.

  • Learn from distinguished guests such as world-renowned NASA scientific illustrator Sally Bensusen
  • Participate in hands-on activities that translate directly into the classroom using a variety of resources, including Smithsonian Science for Global Goals curriculum
  • Tour state of the art facilities such as the Harvard College observatory or get a behind-the-scenes look at Smithsonian museum

The Smithsonian Science Education Center is proud to offer Continuing Education Units (CEUs) for our Academies through Virginia Commonwealth University. The VCU Office of Continuing and Professional Education offers opportunities for personal and professional development as well as custom training solutions, logistical support and skills training in negotiation and mediation. Take the next step at: ocpe.vcu.edu.

* Not all academies are offered every year. Please see here to see which academies will be offered this year. READ MORE

National Council for Science and the Environment

NCSE has established a range of programs to increase the number, quality and diversity of people capable of bringing science to bear on the critical environmental challenges facing our society. These include the:

    EnvironMentors is a science education and national college access program with a mission to mentor and motivate high school students from communities underrepresented in the sciences as they plan and conduct environmental research and acquire skills that will allow them to build careers and become more active stewards of their communities and the environment. EnvironMentors chapters are located around the country and are hosted through partnerships with universities and educational based nonprofit organizations. Since 1992, the program has paired over 2,000 high school students with mentors through its network of chapters throughout the country.

NCSE Alliance of Sustainability and Environmental Leaders The NCSE Alliance of Sustainability and Environmental Academic Leaders (NCSE Leaders’ Alliance) brings together over 100 deans, directors, and academic leaders of environmental and sustainability departments, programs, and schools from all NCSE Members. Participation in the NCSE Leaders’ Alliance provides a valuable peer network for academic leaders to improve the quality and effectiveness of environmental and sustainability higher education programs, research, curricula, and workforce development.

Formerly known as the NCSE Council of Environmental Deans and Directors (CEDD) and the NCSE Community College Alliance for Sustainability Education (CCASE), the NCSE Leaders’ Alliance now captures both CEDD and CCASE and includes the expansion of academic leaders beyond deans and directors as the landscape of sustainability education has continued to evolve and grow. Each NCSE Member is invited to select two deans, directors, and academic leaders to represent their institution on the NCSE Leaders’ Alliance. The NCSE Leaders’ Alliance provides input and perspective on priorities of NCSE Members and advances the quality and effectiveness of interdisciplinary environmental and sustainability education and scholarship.

Current Membership Benefits

  • Participate in the NCSE Alliance of Sustainability and Environmental Academic Leaders (NCSE Leaders’ Alliance), a group for deans, directors, and academic leaders to connect with peers on a range of issues in higher education.
  • Attend the NCSE Annual Conference with select complimentary registrations and unlimited discounted registrations.
  • Engage with local governments and support students to navigate the science-policy interface.
  • Lead and participate in Communities of Practice, technical working groups that support and amplify education, research, and analysis from higher education institutions.
  • Access to NCSE Higher Education Research Reports for analysis of national trends in environmental and sustainability education and research to help institutions innovate.
  • Receive exclusive communications that shares tangible tools for science to engage in environmental decision-making, such as NCSE Pathways and a members-only email list.
  • Attend in-person and virtual events and webinars that convene scientists, thought leaders, and decision-makers.

National Energy Education Development (NEED) Project

Started in 1980, The National Energy Education Development (NEED) Project began as a one-day celebration of energy education when National Energy Education Day was recognized by a Joint Congressional Resolution. In the same year, President Jimmy Carter issued a Presidential Proclamation stressing the need for comprehensive energy education in our schools, a reduction in our dependence of fossil fuels, and increasing energy efficiency and the use of renewable energy technologies. Since its founding 40 years ago, NEED has kept its Kids Teaching Kids philosophy as a fundamental principle of NEED programming – encouraging students to explore, experiment, engage, and encouraging teachers to embrace student leadership in the classroom. NEED trains and assists teachers in harnessing the energy of the classroom – the energy of students.

NEED is expanding and evolving to best meet the needs of both teachers and students – in the classroom and beyond. In just the last decade The NEED Project has grown to encompass a curriculum portfolio of 100+ teacher and student guides designed to engage and teach teachers and students about energy. At the same time, the training opportunities offered by NEED expanded to include a variety of teacher professional development and training for educators and school district energy personnel as well. NEED’s work in after school programs, student clubs, scouting groups, and home school networks also continues to grow.

Growth Energy and National Association of Agricultural Educators (NAAE) High School Biofuels Curriculum

The curriculum is the first industry-supported biofuels curriculum that provides students a guided in-classroom experience and will offer agricultural educators the tools needed to provide students with an array of technical skills and historical knowledge in biofuels.

Agricultural educators considering including this curriculum into their lesson plan for the semester will have access to a number of resources to help supplement the activities provided within the curriculum. These include helpful presentation slides on the history, technology, and policy that make biofuels important for the rural economy, and assessment tools that educators can use to assess and track student progress. Additionally, each activity concludes with a short self-assessment meant to encourage discussion and identify key takeaways that students can reflect on. The curriculum can be downloaded here.

ExploraVision Science Competition K-12

ExploraVision is a science competition that encourages K-12 students of all interest, skill and ability levels to create and explore a vision of future technology by combining their imaginations with the tools of science. All inventions and innovations result from creative thinking and problem solving. That’s what ExploraVision is all about. READ MORE

Ethanol 101

Basic information about ethanol from “Let’s Clear the Air.” READ MORE

University of Idaho 4-H

The University of Idaho’s Biodiesel Education Program has released curriculum designed to help students between the ages of eight and 12 understand the concepts of energy and renewable energy. While the free seven-lesson program was written for 4-H clubs, the Biodiesel Education Program stresses it is also appropriate for use by elementary school teachers.

The curriculum features several hand-on activities, including a matching game, a fossil fuels timeline and a renewable energy model. Other components of the program include an energy tour and viscosity wands.

The program includes two parts, a student workbook and an instructor’s manual. The student workbook contains lessons titled “What is Energy,” “Liquid Fuels as Energy Sources,” “Fossil Fuels,” “Renewable Energy,” “Vegetable Oil and Animal Fat as Sources of Energy,” “How is Biodiesel Made and Used,” and “Scientists and Engineers.” READ MORE and MORE