Attraction of Frankliniella occidentalis Females towards the Aggregation Pheromone Neryl (S)-2-Methylbutanoate and Kairomones in a Y-Olfactometer.

An understanding of insect olfaction allows for more specific alternative methods of pest control. We evaluated the responses of the western flower thrips (WFT, Frankliniella occidentalis) in a Y-olfactometer to estimate gas-phase concentrations of the aggregation pheromone neryl (S)-2-methylbutanoate and known kairomones such as methyl isonicotinate, (S)-(-)-verbenone, and p-anisaldehyde. The gas-phase concentrations of these compounds were obtained from the release rates measured in dynamic headspace cells. The compounds were collected from the headspace using dried solid-phase extraction (SPE) cartridges and analyzed with a triple quadrupole GC-MS/MS. We observed that the aggregation pheromone significantly attracted WFT females at doses of 10 and 100 µg, whereas methyl isonicotinate and p-anisaldehyde significantly attracted WFT females at the highest dose. Verbenone did not produce any significant results. A completely different picture was obtained when the gas-phase concentrations were considered. The minimal gas-phase concentrations of the pheromone required to attract WFT females was 0.027 ng/mL, at least 100 times lower than that of the other two compounds. The relevance and implications of our results are discussed in light of the insect's biology and pest management methods.

Dickkopf-1 directs periosteal bone formation in two murine models of inflammatory arthritis.

OBJECTIVE: The Wnt signalling antagonist Dickkopf-1 (DKK1) inhibits osteoblast differentiation and function and has been described to play a central role in promoting bone loss, while blockade of DKK1 increases bone formation. We investigated the effects of DKK1 on periosteal new bone formation in two murine models of inflammatory arthritis, the antigen-induced arthritis (AIA) and K/BxN serum transfer arthritis (STA) models. METHOD: The flare variant of AIA was induced in wild-type mice and a blocking antibody to DKK1, control rat immunoglobulin G (IgG), or phosphate-buffered saline (PBS) was administered starting on day 14, a time at which inflammation and erosions are known to be established. Knees were assessed for histological inflammation and periosteal new bone formation was quantitated. In addition, STA was generated in transgenic (Tg) mice with osteoblast-specific overexpression of Dkk1 and littermate controls. New bone formation around the wrists of these mice was quantified by micro-computed tomography. RESULTS: Blockade of DKK1 in arthritic mice resulted in significantly more periosteal new bone formation compared to mice treated with control rat IgG or PBS. Conversely, in the setting of increased Dkk1 expression, arthritic Dkk1 Tg mice developed significantly less periosteal new bone than arthritic controls. CONCLUSION: DKK1 is a regulator of periosteal bone formation in inflammatory arthritis. Thus, regulation of DKK1 may be considered as a therapeutic approach in inflammatory diseases in which patients suffer from excessive periosteal bone formation, such as spondyloarthritis.

Total syntheses of spiroviolene and spirograterpene A: a structural reassignment with biosynthetic implications.

The recent natural product isolates spiroviolene and spirograterpene A are two relatively non-functionalized linear triquinane terpenes with a large number of structural homologies. Nevertheless, three significant areas of structural disparity exist based on their original assignments, one of which implies a key stereochemical divergence early in their respective biosyntheses. Herein, using two known bicyclic ketone intermediates, a core Pd-catalyzed Heck cyclization sequence, and several chemoselective transformations, we describe concise total syntheses of both natural product targets and propose that the structure of spiroviolene should be reassigned. As a result, these natural products possess greater homology than previously anticipated.

A role for G protein-coupled receptor 137b in bone remodeling in mouse and zebrafish.

G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba-deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba-/- mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba-deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba-/- fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation.

Asymmetric pyrone Diels-Alder reactions enabled by dienamine catalysis.

Despite the proven value in utilizing pyrone dienes to create molecular complexity via Diels-Alder reactions with varied dienophiles, few examples of effective catalytic, asymmetric variants of this process have been developed. Herein, we show that the use of Jørgensen-Hayashi-type catalysts can convert an array of α,ß-unsaturated aldehydes into chiral dienamines that can formally add in a Diels-Alder fashion to a number of electron-deficient pyrones of the coumalate-type to generate optically active [2.2.2]-bicyclic lactones. In most cases, the reactions proceed with good to excellent diastereo- and enantiocontrol (up to 99% ee). Models to explain that stereoselectivity, as well as several additional transformations of the resultant products, are also presented.

Water vapour and heat combine to elicit biting and biting persistence in tsetse.

BACKGROUND: Tsetse flies are obligatory blood feeders, accessing capillaries by piercing the skin of their hosts with the haustellum to suck blood. However, this behaviour presents a considerable risk as landing flies are exposed to predators as well as the host's own defense reactions such as tail flicking. Achieving a successful blood meal within the shortest time span is therefore at a premium in tsetse, so feeding until replete normally lasts less than a minute. Biting in blood sucking insects is a multi-sensory response involving a range of physical and chemical stimuli. Here we investigated the role of heat and humidity emitted from host skin on the biting responses of Glossina pallidipes, which to our knowledge has not been fully studied in tsetse before. METHODS: The onset and duration of the biting response of G. pallidipes was recorded by filming movements of its haustellum in response to rapid increases in temperature and/or relative humidity (RH) following exposure of the fly to two airflows. The electrophysiological responses of hygroreceptor cells in wall-pore sensilla on the palps of G. pallidipes to drops in RH were recorded using tungsten electrodes and the ultra-structure of these sensory cells was studied by scanning and transmission electron microscopy. RESULTS: Both latency and proportion of tsetse biting are closely correlated to RH when accompanied by an increase of 13.1°C above ambient temperature but not for an increase of just 0.2°C. Biting persistence, as measured by the number of bites and the time spent biting, also increases with increasing RH accompanied by a 13.1°C increase in air temperature. Neurones in wall-pore sensilla on the palps respond to shifts in RH. CONCLUSIONS: Our results show that temperature acts synergistically with humidity to increase the rapidity and frequency of the biting response in tsetse above the levels induced by increasing temperature or humidity separately. Palp sensilla housing hygroreceptor cells, described here for the first time in tsetse, are involved in the perception of differences in RH.

Bacteriological larvicides of dipteran disease vectors.

The apparent success in vector control observed between 1950 and 1970 was followed by worldwide resistance to organosynthetic insecticides wherever they were used intensively. Insect resistance to one or more categories of insecticides has limited the effectiveness of these compounds, and their non-selective mode of action adversely affects non-target organisms. This scenario highlights the need for selective agents in integrated vector control programs. This article gives an overview of the main fundamental and applied research topics on entomopathogenic bacteria in relation to their role in vector control.

Permeabilization of model lipid membranes by Bacillus sphaericus mosquitocidal binary toxin and its individual components.

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of approximately 100-200 pS with long open times and a high open probability. Larger channels (> or =400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (< or =100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity.

The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression.

Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.

Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin.

The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced. Purified crystals were not toxic to Culex pipiens larvae. Comparison of the amino-acid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B. sphaericus had a leucine at this position. Reciprocal site-directed mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine. Native and mutated genes were cloned and overexpressed. Inclusion bodies were tested on C. pipiens larvae. Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity. In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins. In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed. Amino acid 93 of the BinA polypeptide probably belongs to an alpha helix that is sensitive to amino-acid modifications. Position 93 may be a key element in the formation of the BinA-BinB complex responsible for the toxicity and stability of B. sphaericus Bin toxins.

Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena.

Bacteria active against dipteran larvae (mosquitoes and black flies) include a wide variety of Bacillus thuringiensis and B. sphaericus strains, as well as isolates of Brevibacillus laterosporus and Clostridium bifermentans. All display different spectra and levels of activity correlated with the nature of the toxins, mainly produced during the sporulation process. This paper describes the structure and mode of action of the main mosquitocidal toxins, in relationship with their potential use in mosquito and/or black fly larvae control. Investigations with laboratory and field colonies of mosquitoes that have become highly resistant to the B. sphaericus Bin toxin have shown that several mechanisms of resistance are involved, some affecting the toxin/receptor binding step, others unknown.

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.

Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14.

BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction.

[Cloning and expression of the binary toxin genes of Bacillus sphaericus C3-41 in a crystal minus B. thuringiensis subsp. israelensis].

Bacillus sphearicus C3-41, belonging to serotype H5a5b, is ahighly toxic strain isolated from a mosquito-breeding site in China. It has been shown that it had a higher toxicity against Culex spp. than the reference strain 2362 at the laboratory and field conditions. Using synthetic oligonucleotides designed on the basis of the binary toxin gene sequence of 2362, a 1.1 kb DNA fragment was produced and the genetic library was prepared from a HindIII digest of total DNA from C3-41. One colony containing the 3.5 kb HindIII fragment was selected for further studies. Sequence analysis revealed that this 3.5 kb Hind DNA fragment was composed of 3479 nucleic acids and the sequence of the binary toxin gene of C3-41 is completely identical to that of strain 2362. The toxin genes have been transferred into a nontoxic crystal-minus strain of Bacillus thuringiensis subsp. israelensis. The recombinant strains could express the binary toxin of B. sphaericus as crystaline structures during their sporulation. The expression products of the recombinants have a highly toxicity to susceptible Culex pipiens subsp. quinquefasciatus and no toxicity to resistant larvae(Culex pipiens subsp. pipiens) from France.

A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase-cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.

The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae.

BACKGROUND: Following chromosome segregation in anaphase, ubiquitin-dependent degradation of mitotic cyclins contributes to the exit from mitosis. A key step in this process is catalyzed by a ubiquitin-protein ligase known as the anaphase-promoting complex (APC), the regulation of which is poorly understood. The Polo-related protein kinase Cdc5 in Saccharomyces cerevisiae might encode a regulator of the APC, because cdc5 mutant cells arrest with a late mitotic phenotype similar to that observed in cells with defective cyclin destruction. RESULTS: We investigated the role of Cdc5 in the regulation of mitotic cyclin degradation. In cdc5-1 mutant cells, we observed a defect in the destruction of cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC. Overexpression of CDC5 resulted in increased APC activity and mitotic cyclin destruction in asynchronous cells or in cells arrested in metaphase. CDC5 mutation or overexpression did not affect the degradation of the APC substrate Pds 1, which is normally degraded at the metaphase-to-anaphase transition. Cyclin-specific APC activity in cells overexpressing CDC5 was reduced in the absence of the APC regulatory proteins Hct 1 and Cdc20. In G1, Cdc5 itself was degraded by an APC-dependent and Hct1-dependent mechanism. CONCLUSIONS: We conclude that Cdc5 is a positive regulator of cyclin-specific APC activity in late mitosis. Degradation of Cdc5 in G1 might provide a feedback mechanism by which the APC destroys its activator at the onset of the next cell cycle.

Binding of the 51- and 42-kDa individual components from the Bacillus sphaericus crystal toxin to mosquito larval midgut membranes from Culex and Anopheles sp. (Diptera: Culicidae).

Individual components (P51 and P42) from the crystal toxin (Bin) of Bacillus sphaericus were used for in vitro binding competition experiments with brush border membranes (BBMFs) from Culex pipiens and Anopheles gambiae larval midguts. P51 competed for the Bin binding site with a similar affinity to the Bin toxin, on both BBMFs. For C. pipiens, P42 bound non-specifically until P51 was added with maximum binding of P42 at a molar ratio of each component. The binding of P42 was much greater on A. gambiae BBMFs and the presence of either P51 or P42 enhanced the binding of the other component, with highest binding when a mole ratio of each protein was supplied.

Binding kinetics of Bacillus sphaericus binary toxin to midgut brush-border membranes of Anopheles and Culex sp. mosquito larvae.

Direct-binding assays and homologous-competition assays were used to identify specific binding between the radiolabelled toxin of Bacillus sphaericus and brush-border membrane fractions (BBMF) from Anopheles gambiae and Anopheles stephensi, obtained from whole larvae preparations. In both species, the toxin bound to a single class of receptors. BBMF of A. gambiae had the highest binding affinity for the toxin of the species tested, with a dissociation constant (Kd) of 30 +/- 15 nM and a maximum receptor concentration of 5 +/- 1 pmol/mg. Toxin binding to A. gambiae BBMF was compared with that to BBMF from B. sphaericus-susceptible (IP) and B. sphaericus-resistant (SPHAE) Culex pipiens populations. BBMF toxin binding was slower in A. gambiae than in the C. pipiens populations. The BBMF of the B. sphaericus-resistant population of C. pipiens had an association profile that was similar to the susceptible population, despite of the lack of susceptibility in vivo. No relationship between toxicity and irreversibility of toxin binding was detected. On the contrary, toxin dissociation from BBMF was fast and almost complete in BBMF of all species studied.

Resistance to Bacillus sphaericus involves different mechanisms in Culex pipiens (Diptera:Culicidae) larvae.

Field Culex pipiens pipiens (L.) mosquitoes that were collected after a control failure with Spherimos in southern France developed high resistance (> 10,000-fold) to Bacillus sphaericus crystal toxin after < 8 generations of laboratory selection. We show that this resistance is encoded by a single major recessive gene on linkage group I at 22.1 recombination units from the sex locus, and that it is not associated with any loss of binding affinity between brush border membrane fractions and the B. sphaericus radiolabeled toxin. Thus, in Southern France, resistance differs from the high B. sphaericus resistance developed after laboratory selection of Californian C. p. quinquefasciatus. This demonstrates that at least 2 different mechanisms may confer high levels of resistance to B. sphaericus crystal toxin in mosquitoes of the C. pipiens complex. These results have important implications for mosquito control strategies.