Microorganisms for degrading simmondsin and related cyanogenic toxins in jojoba

Summary Four microorganisms that metabolize simmondsin (S) and related cyanogenic toxins from jojoba (Simmondsia chinensis) were isolated by enrichment: Pseudallescheria boydii, a fungus which specifically degrades simmondsin ferulate but not S; Fusarium moniliforme; Flavobacterium aurantiacum; and Pseudomonas maltophilia. The latter three organisms grow on S as a sole carbon and nitrogen source in culture media, but only F. moniliforme attacks S in the complete jojoba meal. Combinations of the four microorganisms at two temperatures, and with free air or limited air exchange for up to 20 days, were tested on jojoba meal to determine an optimum detoxification method. Degradation of toxins was most rapid and complete when Pseudallescheria boydii and Fusarium moniliforme together were incubated on jojoba meal at 25°C with free air exchange for 20 days. Mice were fed fermented meals at 0, 5, 10 and 20% substitution levels to determine detoxification and nutritional quality. Average daily gains during rapid growth of weanling (1–3 weeks) and mature (4–8 weeks) mice did not differ significantly from controls for mice on all diets containing fermented meal. Diets containing fungally detoxified jojoba meal were more efficient for maintaenance of mature weight than jojoba meal detoxified with enzymes naturally present in the meal. Meal can be detoxified by ensilage for 20 days at 80% water content. Detoxification is attributed to as yet unidentified enzymes inherent in the jojoba seed.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentionedOffprint requests to: Thomas P. Abbott     

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, <Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, with a special emphasis on the role of urea synthesis in ammonia detoxification. P. sinensis is ureogenic and possesses a full complement of ornithine-urea cycle enzymes in its liver. It is primarily ureotelic in water, and the estimated rate of urea synthesis in unfed animals was equivalent to only 1.5% of the maximal capacity of carbamoyl phosphate synthetase I (CPS I) in its liver. Approximately 72 h was required for P. sinensis to completely digest a meal of prawn meat. During this period, there were significant increases in ammonia contents in the stomach at hour 24 and in the intestine between hours 12 and 36, which could be a result of bacterial activities in the intestinal tract. However, ammonia contents in the liver, muscle, brain and plasma remained unchanged throughout the 72-h post-feeding. In contrast, at hour 24, urea contents in the stomach, intestine, liver, muscle, brain and plasma increased significantly by 2.9−, 3.5−, 2.6−, 2.9−, 3.4 and 3.0-fold, respectively. In addition, there was a 3.3- to 8.0−fold increase in the urea excretion rate between hours 0 and 36 post-feeding, which preceded the increase in ammonia excretion between hours 12 and 48. By hour 48, 68% of the assimilated N from the feed was excreted, 54% of which was excreted as urea-N. The rate of urea synthesis apparently increased sevenfold during the initial 24 h after feeding, which demanded only 10% of the maximal CPS I capacity in P. sinensis. The postprandial detoxification of ammonia to urea in P. sinensis effectively prevented postprandial surges in ammonia contents in the plasma and other tissues, as observed in other animals, during the 72-h period post-feeding. In addition, postprandial ammonia toxicity was ameliorated by increased transamination and synthesis of certain amino acids in the liver and muscle of P. sinensis. After feeding, a slight but significant increase in the glutamine content occurred in the brain at hour 24, indicating that the brain might experience a transient increase in ammonia and ammonia was detoxified to glutamine.     

An in vitro analysis of intestinal ammonia handling in fasted and fed freshwater rainbow trout (Oncorhynchus mykiss)

Ammonia transport and metabolism were investigated in the intestinal tract of freshwater rainbow trout which had been either fasted for 7 days, or fasted then fed a satiating meal of commercial trout pellets. In vivo, total ammonia concentrations (T _amm) in the chyme were approximately 1 mmol L^?1 across the entire intestine at 24 h after the meal. Highest chyme pH and P _NH3 values occurred in the posterior intestine. In vitro gut sac experiments examined ammonia handling with mucosal (Jm_amm) and serosal (Js_amm) fluxes under conditions of fasting and feeding, with either background (control ≤0.013 mmol L^?1) or high luminal ammonia concentrations (HLA = 1 mmol L^?1), the latter mimicking those seen in chyme in vivo. Feeding status (fasted or fed) appeared to influence ammonia handling by each individual section. The anterior intestine exhibited the greatest Jm_amm and Js_amm values under fasted control conditions, but these differences tended to disappear under typical post-feeding conditions when total endogenous ammonia production (Jt_amm = Js_amm ? Jm_amm, signs considered) was greatly elevated in all intestinal sections. Under fasted conditions, glutamate dehydrogenase (GDH) and glutaminase (GLN) activities were equal across all sections, but the ammonia-trapping enzyme glutamine synthetase (GS) exhibited highest activity in the posterior intestine, in contradiction to previous literature. Feeding clearly stimulated the total rate of endogenous ammonia production (Jt_amm), even in the absence of a high luminal ammonia load. This was accompanied by an increase in GDH activity of the anterior intestine, which was also the site of the largest Jt_amm. In all sections, during HLA exposure, either alone or in combination with feeding, there were much larger increases in endogenous Jt_amm, most of which was effluxed to the serosal solution. This is interpreted as a response to avoid potential cytotoxicity due to overburdened detoxification mechanisms in the face of elevated mucosal ammonia. Thus T _amm of the intestinal tissue remained relatively constant regardless of feeding status and exposure to HLA. Ammonia production by the gut may explain up to 18 % of whole-body ammonia excretion in vivo under fasting conditions, and 47 % after feeding, of which more than half originates from endogenous production rather than from absorption from the lumen.     

Direct and indirect competitive monoclonal antibody-based ELISA of Aflatoxin B1 in groundnut

Direct and indirect competitive enzyme-linked immunosorbent assays were optimized for the determination of aflatoxin B₁ in groundnut utilizing a specific monoclonal antibody developed at the University of Strathclyde, UK. The monoclonal antibody was conjugated to horseradish peroxidase (HRP) for direct competitive assay, while a commercially available goat-antimouse IgG-HRP conjugate was employed for indirect competitive ELISA. Both ELISAs detected aflatoxin B₁ as low as 20 pg/well. Methanol-water-KCl (70 + 30 v/v, 0.5 %) extracts of groundnut were assayed by ELISA after diluting 1: 10 with PBS-Tween buffer or subjected to simple cleanup for 5:1 concentration prior to assay. The mean recoveries from groundnut spiked with 10 to 200/ig/kg of pure aflatoxin B₁ were >90% in either ELISA, but the toxin recoveries at concentrations of 1–5μg/kg were only 65–67 % when subjected to cleanup and concentration before assay. The mean within-assay, inter-assay, and sub-sample coefficients of variation by ELISA of aflatoxin B₁ in naturally contaminated groundnuts were, respectively, 8.9%, 11.1%, and 7.9% for direct competitive assay and 4.6%, 11.2%, and 8% for indirect competitive assay. Both ELISA methods are useful for routine analysis of aflatoxin B₁ in groundnuts.     

Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis

Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)‐chelating peptides and by sequestering PC–metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As–PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC₂–Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC₂–Cd transport activity compared with PC₂–As. In contrast, barley vacuoles readily accumulated comparable levels of PC₂–Cd and PC₂–As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC‐type transporters. Interestingly, barley vacuoles exhibited enhanced PC₂ transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non‐essential toxic metal(loid)s.     

Sedimentation characteristics and effect of molasses concentration and medium supplementation on the ethanol productivity of an ethanol tolerant palm wineSaccharomyces

Summary Sedimentation rates ranging from 58.82 to 93.10% were recorded for six palm wineSaccharomyces yeast isolates. Isolates S, J and A₃, gave values of 90.00, 91.95 and 93.10% respectively compared to 81.54% for a standard lager beer yeast. Fermentation of unclarified molasses medium 10–30° Brix with isolated J gave ethanol productivity of 74.35-67.07%. Soyabean, groundnut or castor oil seed meal supplements significantly enhanced ethanol productivity in all the molasses media.     

Induced systemic resistance in groundnut by foliar application of Headline (Pyraclostrobin 20% WG)

Plant’s own defence mechanisms can be stimulated by the application of elicitors for its protection. We evaluated the ability of Headline (Pyraclostrobin 20% WG) to induce systemic resistance in 40-day-old field-grown groundnut plants by foliar application. Elevated levels of defence-related enzymes such as phenylalanine ammonia lyase, peroxidase, β 1,3 glucanase, nitrate and nitrite reductase and other biochemical parameters such as total phenol, total protein, total carbohydrate and total chlorophyll content were found highest in Headline treated plants on fourth day after treatment in comparison with untreated plants. The treated plants showed enhanced growth and development as well as increased yield up to 16%. About 10% mortality rate was found in groundnut seeds treated with Headline when grown on Sclerotium rolfsii-infested soil than control. These results indicate that foliar application of Headline can be successfully developed the broad-spectrum resistance against pathogens in groundnut.     

Sophorolipid production from delignined corncob residue by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and Cryptococcus curvatus ATCC 96219

Delignined corncob residue hydrolysate (DCCRH) and detoxified DCCRH were used for single cell oil (SCO) and single cell protein (SCP) production of Cryptococcus curvatus ATCC 96219 and for sophorolipid (SL) production of Wickerhamiella domercqiae var. sophorolipid CGMCC 1576. Both C. curvatus and W. domercqiae could utilize glucose in DCCRH to grow and accumulate lipids or particle-shaped SLs. DCCRH detoxification by activated carbon adsorption not only improved cell growth and lipid accumulation of C. curvatus but also increased SL production and proportion of lactonic SL in total SL. A total biomass of 17.36 g/l with a lipid content of 44.36 % could be achieved after cultivation of C. curvatus on the detoxified DCCRH. The predominant fatty acids of the produced SCO were oleic, stearic, and palmitic acids (27.2, 20.5, and 15.7 %, respectively). When W. domercqiae cells were cultivated on DCCRH and SCO, total SL production of 39.08 g/l (DCCRH?+?SCO) and 42.06 g/l (detoxified DCCRH?+?SCO) were obtained. Furthermore, when cell lysate of C. curvatus, oleic acid, and DCCRH/detoxified DCCRH was used as nitrogen and carbon sources, total SL production reached 37.19 g/l and 48.97 g/l, respectively. These results demonstrated that renewable DCCRH can be utilized for the production of high-value SCO and SLs.     

Host range and some properties of groundnut rosette virus

Two isolates of groundnut rosette virus from East Africa (GRVE₁ and GRVE₂) and from West Africa (GRVW₁ and GRVW₂) were transmitted by Aphis craccivora obtained from West Africa. A third isolate from West Africa (GRVW₃) was not transmitted by A. craccivora from three widely separated sources. GRVW₁, GRVW₂ and GRVW₃ caused leaf-symptoms in groundnut of a mosaic pattern in light and dark green. GRVE₁ and GRVE₂ caused chlorosis or chlorosis and leaf distortion as well as mosaic symptoms. Groundnut plants with GRVW₁ could not be infected by means of aphids with GRVE₁, and GRVE₁ gave similar protection against GRVW₁, which suggests that they are strains of the same virus. All isolates were transmissible manually from groundnut to groundnut (Arachis hypogea), Trifolium incarnatum and T. repens, and caused systemic infection. Inoculated Nicotiana clevelandii and N. rustica developed symptoms but virus could not be recovered from them. Chenopodium amaranticolor, C. hybridum and C. quinoa showed local lesions on inoculated leaves. Virus could be acquired by aphids from groundnut or Trifolium repens infected by means of aphids, but not from those infected by manual inoculation. Virus could not be recovered from T. incarnatum manually or by aphids, but was transmitted by cleft-grafting from clover to groundnut. Saps extracted in borax buffer plus zinc sulphate at pH 9 from plants infected with GRVW₁ and GRVE₁ remained infective at 18° C. for 1 week, and at — 20° C. for up to 4 weeks. Virus could be recovered from frozen leaves. Buffered saps lost infectivity when heated above 50° C. for 10 min.; most were still infective when diluted 1/10 and some at 1/100. Electron micrographs of partially purified preparations contained spherical particles 25–28 mμ in diameter. There were usually only about five per microscope field and they resembled those of some other viruses.     

Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O135 bound to BSA-protein carrier

O135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M _w ∼ 197,000 Da) and the lipopolysaccharide fragment (M _w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.     

Interaction of medium detoxification/supplementation and cell recycling in fermentative xylitol production

Abstract

To study the importance and interactions of pH control and delignification of a medium derived from oat hull hemicellulose, as well as the influence of supplemental nitrogen source and cell recycling, Candida guilliermondii was used as the biocatalyst in repeated batch bioconversion processes in three successive batches each lasting 144 h. The research study was conducted based on a factorial design with three factors consisting of: a) pH control (pH_s: pH constant during the process; pH_i: pH adjusted to 6.0 at the beginning of bioconversion with no control thereafter; and pH_t: pH set to 6.0 before sterilization of the medium with no control thereafter); b) medium detoxification/supplementation (Conc.: medium used directly with no treatment; AC1.25: medium treated with 1.25% activated carbon; AC2.5: medium treated with 2.5% activated carbon; AC5: medium treated with 5% activated carbon; and AC2.5-N: medium detoxified with 2.5% activated carbon and then supplemented with ammonium sulphate); and c) cell recycling [three cycles (C₁, C₂, and C₃)]. Results from the bioconversion process indicated that detoxification of the medium was the least important factor affecting product yield and productivity, while cell recycling and medium supplementation were much more important parameters which needed to be considered to achieve a successful process. Results also indicated that medium supplementation by inorganic nitrogen (ammonium sulphate) could be a requirement to achieve a consistent process performance in consecutive cycles of bioconversion using recycled biocatalyst. However, there is no need to use a supplemental nitrogen source in a single-cycle process.     

Further properties of peanut clump virus and studies on its natural transmission

Purified preparations of particles of peanut clump virus (PCV) had A₂₆₀/A₂₈₀ values (corrected for light scattering) of 1.00. They contained rod-shaped particles with sedimentation coefficients of 183 S and 224 S, and a density in CsCl of 1.32 g/ml. PCV infected 36 species in 8 plant families. No serological relationship was detected between PCV and barley stripe mosaic, beet necrotic yellow vein, Nicotiana velutina mosaic and tobacco mosaic viruses. PCV was seed-borne for two generations in groundnut (Arachis hypogaea) but was not seed-borne in great millet (Sorghum arundinaceum), Phaseolus mungo or Nicotiana benthamiana. Seedlings of groundnut, great millet and wheat (Triticum aestivum) became infected when grown in soil from groundnut fields with outbreaks of clump disease, and the infectivity of soil survived air-drying at 25°C for 3 months. Groundnut seedlings became infected when grown in sterilised soil contaminated with washed roots of naturally-infected S. arundinaceum but not in soil to which roots of naturally infected groundnut or shoots of infected groundnut were added, or in which mechanically inoculated groundnut seedlings were grown at the same time. The patchy distribution of PCV in a crop was related to the infectivity of the soil for groundnut and to the presence of Polymyxa graminis resting spores which could be detected in the roots of S. arundinaceum bait seedlings, but not in those of groundnut. The results indicate that PCV is transmitted by a vector that is resistant to air-drying and closely associated with S. arundinaceum roots. For these reasons P. graminis is thought to be the vector of PCV.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Solid-state fermentation production of tetracycline by <Emphasis Type="Italic">Streptomyces</Emphasis> strains using some agricultural wastes as substrate

The ability of Streptomyces sp. OXCI, S. rimosus NRRL B2659, S. rimosus NRRL B2234, S. alboflavus NRRL B1273 S. aureofaciens NRRL B2183 and S. vendagensis ATCC 25507 to produce tetracycline using some local agricultural wastes as solid state media, were assessed. The wastes employed include peanut (groundnut) shells, corncob, corn pomace and cassava peels. Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity. All the strains produced tetracycline in a solid-state fermentation process containing peanut (groundnut) as the carbohydrate source. Streptomyces sp. OXC1 had the highest ability for tetracycline production with peanut shells as the substrate in solid fermentation (13.18 mg/g), followed by S. vendagensis ATCC 25507 (11.08 mg/g), S.rimosus NRRL B1679 (8.46 mg/g), S. alboflavus NRRL B1273 (7.59 mg/g), S. rimosus NRRL B2234 (6.37 mg/g), S. aureofaciens NRRL B2183 (4.27 mg/g). Peanut (groundnut) shells were the most effective substrate (4.36 mg/g) followed by corncob (2.64 mg/g), cassava peels (2.16 mg/g) and corn pomace (1.99 mg/g). The composition of the peanut (groundnut) shell medium optimal for tetracycline production were peanut shells 100 g, organic nitrogen (peanut meal) 10 g, (NH ₄)₂ SO₄ 1 g, KH₂ PO ₄ 0.5 g, CaCO₃ > 0.5 g, NaCl 0.5 g, MgSO₄ · 7H₂ O 0.5 g, soluble starch 10 g, peanut oil 0.25 ml with initial moisture content of 65–68%, and initial pH 5.3–6.3. Substrate (1 g dry weight) was inoculated with 1.0 × 10 ⁸ conidia per ml and incubated at 28–31 °C for 5–7 days, producing 13.18 mg/g of total tetracycline. Tetracycline detection started on day 3 and attained its maximum level on day 5.     

Factors Affecting the Acetylene-Induced Decline during Nitrogenase Assays in Root Nodules of Myrica gale L

Our goal was to determine why the rate of acetylene reduction by nodules of actinorhizal plants declines after an initial peak value. The decline was eliminated by pretreatment with argon, indicating that the decline is initiated by cessation of ammonia synthesis. When O₂ concentration was decreased during the decline, the rate of acetylene reduction increased. This shows that during the decline there is either O₂ toxicity or competition between respiration and nitrogenase for reductant. The decline was not eliminated when uptake hydrogenase was inactivated by pretreatment with acetylene, showing that cessation of H₂ oxidation is not the primary cause of the decline. The effects of a variety of other treatments on the decline were also studied. Overall, we conclude that the cessation of ammonia formation is the primary cause of the acetylene-induced decline. We hypothesize that the supply of reductant for nitrogenase depends on amino acids that are depleted following cessation of ammonia formation. We also conclude that the initial peak rate of acetylene reduction provides the best measure of nitrogenase activity.     

A dry granular inoculant of Rhizobium for soil application

A dry granular inoculant of Rhizobium was prepared from sodium alginate and peralite. High numbers of two groundnut (Arachis hypogaea) Rhizobium strains, NC 92 and TAL 1000 used to prepare inoculants survived in dry granules beyond 180 days. The viable counts were 9.72 and 9.91 log₁₀ rhizobia g^-1 of dry granules for NC 92 and TAL 1000, respectively compared to 8.0 log₁₀ rhizobia g^-1 of peat inoculant for NC 92 at the end of six months storage. The granular inoculant was free from contaminants. In a pot culture experiment the granular inoculant applied to the soil gave similar results when seeds were dressed with a peat inoculant; nodulation and growth of groundnut were similar. The major advantage of this inoculant is that, it can be stored in a dry state without losing much viability.     

In Vitro Detoxification of Aflatoxin B1, Deoxynivalenol,Fumonisins, T-2 Toxin and Zearalenone by Probiotic Bacteria from Genus Lactobacillus and Saccharomyces cerevisiae Yeast

The aim of the following research was to determine the detoxification properties of probiotic Lactobacillus sp. bacteria (12 strains) and S. cerevisiae yeast (6 strains) towards mycotoxins, such as aflatoxin B₁, deoxynivalenol, fumonisins, T-2 toxin and zearalenone, which pose as frequent feed contamination. The experiment involved analysing changes in concentration of mycotoxins in PBS solutions, after 6, 12 and 24 h of incubation with monocultures of tested microorganisms, measured by high-performance liquid chromatography (HPLC). We found that all strains detoxified the mycotoxins, with the highest reduction in concentration observed for the fumonisin B₁ and B₂ mixture, ranging between 62 and 77% for bacterial strains and 67–74% for yeast. By contrast, deoxynivalenol was the most resistant mycotoxin: its concentration was reduced by 19–39% by Lactobacillus sp. strains and 22–43% by yeast after 24 h of incubation. High detoxification rates for aflatoxin B₁, T-2 toxin and zearalenone were also observed, with concentration reduced on average by 60%, 61% and 57% by Lactobacillus, respectively, and 65%, 69% and 52% by yeast, respectively. The greatest extent of reduction in the concentration for all mycotoxins was observed after 6 h of incubation; however, a decrease in concentration was noted even after 24 h of incubation. Thus, the tested microorganisms can potentially be used as additives to decrease the concentrations of toxins in animal feed.

     

Cloning and functional expression of Rh50-like glycoprotein, a putative ammonia channel, in Aedes albopictus mosquitoes

Evidence has shown that female mosquitoes can deaminate more than 80% of the ingested bloodmeal protein amino acids, and thus lead to a massive amount of ammonia production. Ammonia transport is a critical step for detoxifying ammonia in organisms. Here we characterized a putative ammonia channel gene, Rhesus (Rh) 50 glycoprotein, from Aedes albopictus (AalRh50) and determined the difference of its expression profile in different tissues at both message and protein levels as well as its response to a blood meal. We showed that AalRh50 shares a low identity with E. coli ammonia transporter (EcoAmtB), but higher identities with human RhBG and Drosophila Rh50 genes. The analysis of ammonia-conductance sites indicates that AalRh50 has residue substitutions of S237L (equivalent to S219 in AmtB) in the external vestibule, F127I (equivalent to F107 in AmtB) in the pore entrance, and S281N (equivalent to S263 in AmtB) in the internal vestibule, which could alter or reduce ammonia-conductance activity. The results from quantitative real-time-PCR and immunohistochemistry revealed that AalRh50 is expressed at significantly higher levels in the head, Malpighian tubules, and thorax of the non-blood-fed females, suggesting that AalRh50 might play roles in maintaining normal neurotransmitter metabolism, acid-base balance, and flight energy production in different tissues of mosquitoes at the non-blood-fed condition. A blood meal significantly increases AalRh50 expression in midgut, fat body, and Malpighian tubules from 3 or 6 to 24 h post feeding, indicating that AalRh50 plays an important role in detoxification of excess systemic ammonia of female adults during the gonotrophic cycle.     

Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica

Information on the effects of elevated ammonia on invertebrates in general, and polar Mollusks in particular, is scant. Questions of ammonia sensitivity are interesting for several reasons, particularly since predicted global change scenarios include increasing anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species are often lipid-rich, and authors have speculated that there is a linkage between elevated levels of lipids/trimethylamine oxide and enhanced ammonia tolerance. In the present study, we sought to examine ammonia tolerance and effects of elevated exogenous ammonia on several key aspects of the physiology and biochemistry of the pteropod mollusk, Clione limacina antarctica. We determined that the 96-h LC₅₀ value for this species is 7.465?mM total ammonia (Upper 95% CL?=?8.498?mM and Lower 95% CL?=?6.557?mM) or 0.51?mg/L as unionized ammonia (NH₃) (at a pH of 7.756). While comparative data for mollusks are limited, this value is at the lower end of reported values for other species. When the effects of lower ammonia concentrations (0.07?mM total ammonia) on oxygen consumption and ammonia excretion rates were examined, no effects were noted. However, total ammonia levels as low as 0.1?mM (or 0.007?mg/l NH₃) elevated the activity of the ammonia detoxification enzyme glutamine synthetase by approximately 1.5-fold. The values for LC₅₀ and observable effects on biochemistry for this one species are very close to permissible marine ammonia concentrations, indicating a need to more broadly determine the sensitivity of zooplankton to potential elevated ammonia levels in polar regions.