Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.     

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma. The improvement of the protective effect of BCG vaccine mediated by a DNA-HSP65 booster suggests that our strategy may hold promise as a safe and effective vaccine against TB.     

Ecological differences between hamlet (Hypoplectrus: Serranidae) colour morphs: between-morph variation in diet

Dietary differences between hamlet Hypoplectrus spp. colour morphs were examined in fishes from Puerto Rico, U.S. Virgin Islands, Curacao, Honduras and Belize. Hamlet diet across all countries was characterized by large overlap between most colour morphs in both the proportion and numbers of dietary items consumed, although some differences between morphs were apparent. Indigo hamlets Hypoplectrus indigo were the only morph to consume fishes (blue chromis Chromis cyanea and sunshinefish Chromis insolata ) almost exclusively. The sympatric occurrence of other ecologically indistinguishable colour morphs, however, suggests that divergent ecological selection alone cannot explain population divergence in hamlets. Geographical variation in diet was also observed within black Hypoplectrus nigricans and yellowtail Hypoplectrus chlorurus hamlets which may reflect geographical differences in prey availability or differences in prey choice.     

The role of heterogenous environmental conditions in shaping the spatiotemporal distribution of competing Aedes mosquitoes in Panama: implications for the landscape of arboviral disease transmission

Monitoring the invasion process of the Asian tiger mosquito Aedes albopictus and its interaction with the contender Aedes aegypti, is critical to prevent and control the arthropod-borne viruses (i.e., Arboviruses) they transmit to humans. Generally, the superior ecological competitor Ae. albopictus displaces Ae. aegypti from most geographic areas, with the combining factors of biology and environment influencing the competitive outcome. Nonetheless, detailed studies asserting displacement come largely from sub-tropical areas, with relatively less effort being made in tropical environments, including no comprehensive research about Aedes biological interactions in Mesoamerica. Here, we examine contemporary and historical mosquito surveillance data to assess the role of shifting abiotic conditions in shaping the spatiotemporal distribution of competing Aedes species in the Republic of Panama. In accordance with prior studies, we show that Ae. albopictus has displaced Ae. aegypti under suboptimal wet tropical climate conditions and more vegetated environments within the southwestern Azuero Peninsula. Conversely, in the eastern Azuero Peninsula, Ae. aegypti persists with Ae. albopictus under optimal niche conditions in a dry and more seasonal tropical climate. While species displacement was stable over the course of two years, the presence of both species generally appears to fluctuate in tandem in areas of coexistence. Aedes albopictus was always more frequently found and abundant regardless of location and climatic season. The heterogenous environmental conditions of Panama shape the competitive outcome and micro-geographic distribution of Aedes mosquitoes, with potential consequences for the transmission dynamics of urban and sylvatic zoonotic diseases.

     

Two levels of resting potential in cardiac purkinje fibers

In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was “N-shaped,” and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride.     

Hydrogen sulfide effects on stomatal apertures

Hydrogen sulfide (H₂S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H₂S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H₂S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H₂S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsicum anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.Key words: abscisic acid, GYY4137, hydrogen sulfide, nitric oxide, stomatal apertureThe effects of hydrogen sulfide on plants have been studied for many years, but it is only recently that it has been suggested that this gas is acting as a signaling molecule. In animals this has been well established^1,2 and it has been suggested that H₂S be grouped together with other gasotransmitters.^2,3 This group will also contain nitric oxide (NO) which as well as having established roles in animals is also known to cause stomatal closure in plants.^4,5 With this in mind, we previously investigated whether H₂S may also have an effect on stomatal closure, using a model organism Arabidopsis thaliana.⁶ The study used two different H₂S donors, sodium hydrosulfide (NaSH) and morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithionate (GYY4137). The former will release H₂S in an instant burst which soon dissipates, which questions the wisdom of its use. GYY4137 on the other hand will release H₂S much more slowly and in a manner which is more likely to reflect physiological generation of H₂S.^7,8 Both donors caused stomatal that had previously been exposed to light to open even further. If leaf tissues were not light treated H₂S compounds once again caused stomata to open. Furthermore, H₂S treatment prevented stomatal closure caused by dark treatment. To investigate the possible mechanism of this effect, tissues were treated with the plant hormone abscisic acid (ABA) to initiate NO generation and then NO accumulation was measured in the absence and presence of H₂S donors using fluorescent probes and confocal microscopy.⁹ Both NaSH and GYY4137 caused a reduction in the accumulation of NO. This suggests that H₂S may be acting by a disruption of NO signaling, which results in the alteration of guard cell physiology.Others have reported different effects of H₂S on stomatal movements. Garcia-Mata and Lamattina¹⁰ found that both H₂S donors NaSH and GY4137 caused stomatal closure in different plant species including Vicia faba, Arabidopsis thaliana and Impatiens walleriana. Use of glibenclamide, which is an ABC transport inhibitor, reduced the effect. Cystathione γ lyase and L-Cys desulfhydrase are enzymes which may be responsible for H₂S synthesis and stomatal movements were also reduced by propargylglycine, an inhibitor of these enzymes. It was suggested therefore that H₂S helps to mediate ABA signaling pathway in guard cells. This paper was further discussed following its publication by Desikan.¹¹ However, this seems to be in conflict with the work we reported. This would not be the first time that there has been contradictory data when it comes to reporting stomatal movements, as ethylene has been shown to mediate auxin-induced opening¹² and to cause stomatal closure.¹³More recently it has been reported that stomatal conductance was increased by carbonyl sulfide (COS).¹⁴ The authors went on to suggest that this effect was mediated by H₂S which was produced from COS hydrolysis. This seems to support our original data. Therefore, here we report on the effects of both NaSH and GYY4137 on a different plant species and one which has relevance as an important crop, that is Capsicum anuum. GYY4137 was supplied as in our previous paper in reference ⁶ and ⁷. As can be seen in Figure 1A NaSH caused stomata to open further, even though the leaf tissue had been exposed to the light. Stomata were able to close, as ABA treatment demonstrated, therefore showing that the stomata were not defective. When the experiments were repeated with GYY4137 (Fig. 1B) and smaller but similar effect of the addition of the H₂S donor was seen. This would be expected as the release of H₂S from GYY4137 would be slower and more prolonged than from NaSH.^7,8 To investigate if NO accumulation is also effected in Capsicum when treated with H₂S donors, leaf tissue was treated with ABA to initiate NO generation and NO measured by the use of DAF2-DA as previously reported in references ⁶ and ⁹. Once again the presence of H₂S donors dramatically reduced the amount of NO that was measured following ABA treatment (Fig. 2). This once again suggests that H₂S is having an effect on NO metabolism which may account for the stomata aperture measurements. It has been suggested in animal systems that H₂S and NO react, resulting in the formation of nitrosothiols/nitrothiol-like species¹⁵ which could have signaling effects in their own right. NO in plants has been reported to lead to increased cGMP and/or increased nitrosylation of proteins,⁵ but if H₂S was removing the bioavailability of NO both mechanisms are likely to be reduced.Open in a separate windowFigure 1H₂S donors cause stomatal opening in Capsicum anuum. The leaves of analyzed from Capsicum anuum plants which were between 6 and 7 weeks old. Stomatal bioassays were performed as described previously by Desikan et al.⁹ Epidermal peels were incubated in MES-KCl buffer [10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KCl, 50 µM CaCl₂, pH 6.15] for 2.5 h exposed to the direct lightning (in 60–100 IE m⁻² s⁻¹) before the addition of various compounds. (A) Samples were sheltered from direct lighting and treated with ABA or NaHS for 2.5 h and left under the day light conditions before stomata apertures were analyzed. (B) Samples were sheltered from direct lighting and treated with ABA or GYY 4137 for next 2 h and left under the day light conditions before stomata apertures were analyzed. Apertures were measured using a light microscope and imaging camera with LEICA QWIN image processing and analysis software (Leica Microsystems and Imaging Solutions, Cambridge, UK). n = 40 stomatal apertures, ±SE. GYY4137 was synthesis as previously described in reference ⁷.Open in a separate windowFigure 2H₂S donors reduce NO accumulation in Capsicum anuum. Nitric oxide accumulation was estimated using the specific NO dye DAF2-DA (Calbiochem, Nottingham, UK), using the method described previously by Desikan et al.⁹ Epidermal fragments in MES-KCl buffer (10 mM MES, 5 mM KCl, 50 µM CaCl₂, pH 6.15) were exposed to the direct lightning for 2 h. After 2 h samples were loaded with 30 µM DAF2-DA for 15 min before washing with MES-KCl buffer; three times for 10 min. Fragments were subsequently incubated for a further 30 min in the presence of various compounds (as indicated below) before images were visualized using CLSM (excitation 488 nm, emission 515 nm; Nikon PCM2000, Kingston-upon-Thames, UK). Images acquired were analyzed using SCION IMAGE software (Scion, Frederick, MD, USA). (A) Control with no treatment; (B) ABA (50) treatment; (C) NaHS (100 µm) treatment alone; (D) ABA treatment in the presence of NaHS; (E) GYY4137 (100 µm) treatment alone; (F) ABA treatment in the presence of NaHS.NO metabolism is involved in a wide range of plant functions, including seed germination,¹⁶ floral development,¹⁷ root gravitropism¹⁸ and gene expression¹⁹ as well as controlling stomatal function.⁴ H₂S on the other hand may be present in or around plants for a variety to reasons. H₂S can be produced endogenously by for example by plastid located cysteine desulfhydrases,²⁰ or H₂S may come from the environment,²¹ including the soil and waters.²² This is further discussed in a recent review in reference ²³. Therefore future work should be focused on the interplay between H₂S from a variety of sources on the NO metabolism of a range of plant tissues. Not all affects of H₂S will be mediated by NO, with alterations of glutathione on H₂S treatment being reported for example.²⁴ But the full extent of the modulation of NO accumulation and signal by both exogenous and endogenous H₂S needs to be explored so the role of these gasotransmitters^2,3 in mediating hormone and stress responses in plants can be fully understood.     

Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels

Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway.     

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

Sub-chronic exposure to 1,1-dichloropropene induces frameshift mutations in lambda transgenic medaka

1,1-Dichloropropene (1,1-DCP) is a contaminant present in both ground and surface waters used as sources for drinking water. Structural similarity to several compounds with known mutagenicity and carcinogenicity, and recent demonstration of mutagenicity in vitro, suggest this compound may be similarly mutagenic in vivo. A transgenic fish model, the lamda transgenic medaka, was used to evaluate the potential mutagenicity of this contaminant in vivo following sub-chronic exposure for 6 weeks. Mutant frequencies of the cII target gene (MF) increased six-fold in the livers of fish exposed to the lowest 1,1-DCP exposure concentration (0.44 mg/L, MF = 18.4 x 10(-5), and increased with each treatment, culminating in a 32-fold induction in fish from the highest 1,1-DCP treatment (16.60 mg/L, MF = 96.3 x 10(-5). Mutations recovered from treated fish showed a distinctive mutational spectrum comprised predominantly of +1 frameshift mutations, induced 166-fold above that of untreated animals. The majority of frameshifts were +1 insertions at thiamine and adenine. These results represent the first evidence of mutagenicity of 1,1-DCP in vivo, and of the highly characteristic spectrum of induced mutations dominated by +1 frameshift mutations. Based upon results from previous in vitro studies, the similar role of glutathione S-transferase (GSTT1-1) in the activation of 1,1-DCP to a mutagen in vivo is also suggested. This study further illustrates the utility of the lamda transgenic medaka as a model for identifying and characterizing potential genetic health risks associated with chemical exposures in the environment.