Both farnesyl diphosphate synthase genes are involved in the production of alarm pheromone in the green peach aphid Myzus persicae

Farnesyl diphosphate synthase (FPPS) catalyzes the formation of FPP, providing the precursor for the biosynthesis of (E)‐β‐farnesene (EβF) in plants, but it is unknown if FPPS supplies the precursor for the biosynthesis of EβF, the major component of aphid alarm pheromone, though our previous studies support the hypothesis that EβF is synthesized by the aphid itself. Here, we used two cohorts of the green peach aphid Myzus persicae separately, reared on pepper plant and artificial diet to test the correlations among droplet emission, EβF quantity, and FPPS gene expression. It was found that the proportion of aphids emitting cornicle droplets and the quantity of EβF per milligram of aphid were both significantly different between the two cohorts, which were positively correlated with the expression of the two FPPS genes ( MpFPPS1/ 2) in M. persicae. These results were further confirmed by RNAi‐mediated knockdown of MpFPPS1/ 2. Specifically, knockdown of MpFPPS1/ 2 imposed no significant cost on the survival of aphid but remarkably increased the number of offspring per aphid; most importantly, knockdown of MpFPPS1/ 2 significantly reduced the proportion of aphids emitting droplets and the quantity of EβF calculated as per the weight of aphid. Our results suggest that both FPPS genes are involved in the production of EβF in M. persicae and cornicle droplet emission is closely associated with the EβF release in the aphid.     

Effect of synthetic and plant‐extracted aphid pheromones on the behaviour of Aphidius colemani

Parasitoid females use several chemical cues to locate hosts. A better knowledge of how they respond to a complex of these cues in a small range may help us to understand how to manipulate the parasitoids in the field. Here, the response of the aphid parasitoid Aphidius colemani to a mixture of odours of synthetic and plant‐extracted nepetalactone (a component of aphid sex pheromone) and to (E)‐β‐farnesene (aphid alarm pheromone) was investigated. The behavioural responses of A. colemani to three semiochemical groups with different concentrations were studied in a square arena. Parasitoid females were significantly attracted by the semiochemicals, when their concentrations were high, in which case the females spent more time in squares with semiochemicals. The majority of females preferred plant‐extracted nepetalactone, when it was in high concentration, but they consistently did not respond to (E)‐β‐farnesene.     

Alarm pheromone mediates production of winged dispersal morphs in aphids

The aphid alarm pheromone ( E )- β -farnesene (EBF) is the major example of defence communication in the insect world. Released when aphids are attacked by predators such as ladybirds or lacewing larvae, aphid alarm pheromone causes behavioural reactions such as walking or dropping off the host plant. In this paper, we show that the exposure to alarm pheromone also induces aphids to give birth to winged dispersal morphs that leave their host plants. We first demonstrate that the alarm pheromone is the only volatile compound emitted from aphid colonies under predator attack and that emission is proportional to predator activity. We then show that artificial alarm pheromone induces groups of aphids but not single individuals to produce a higher proportion of winged morphs among their offspring. Furthermore, aphids react more strongly to the frequency of pheromone release than the amount of pheromone delivered. We suggest that EBF leads to a 'pseudo crowding' effect whereby alarm pheromone perception causes increased walking behaviour in aphids resulting in an increase in the number of physical contacts between individuals, similar to what happens when aphids are crowded. As many plants also produce EBF, our finding suggests that aphids could be manipulated by plants into leaving their hosts, but they also show that the context-dependence of EBF-induced wing formation may hinder such an exploitation of intraspecific signalling by plants.     

Expressing an (E)-β-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid

(E)-β-Famesene(EβF) synthase catalyses the production of EβF,which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies.Many plants possess EβF synthase genes and can release EβF to repel aphids.In order to effectively recruit the plant-derived EβF synthase genes for aphid control,by using chloroplast transit peptide(CTP) of the small subunit of Rubisco(rbcS) from wheat(Triticum aestivum L.),we targeted AαβFS1,an EβF synthase gene from sweet wormwood(Artemisia annua L),to the chloroplast of tobacco to generate CTP + AαβFS1 transgenic lines.The CTP +AαβFS1 transgenic tobacco plants could emit EβF at a level up to 19.25 ng/day per g fresh tissues,4-12 fold higher than the AαβFS1 transgenic lines without chloroplast targeting.Furthermore,aphid/parasitoid behavioral bioassays demonstrated that the CTP + AαβFS1 transgenic tobacco showed enhanced repellence to green peach aphid(Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae,thus affecting aphid infestation at two trophic levels.These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EβF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.     

Cues of Predation Risk Induce Instar‐ and Genotype‐Specific Changes in Pea Aphid Colony Spatial Structure

Deploying collective antipredator behaviors during periods of increased predation risk is a major determinant of individual fitness for most animal groups. Pea aphids, Acyrthosiphon pisum, which live in aggregations of genetically identical individuals produced via asexual reproduction warn nearby conspecifics of pending attack by secreting a volatile alarm pheromone. This alarm pheromone allows clone‐mates to evade predation by walking away or dropping off the host plant. Here, we test how a single alarm pheromone emission influences colony structure and defensive behavior in this species. Relative to control colonies, groups exposed to alarm pheromone exhibited pronounced escape behavior where many individuals relocated to adjacent leaves on the host plant. Alarm pheromone reception, however, also had subtle instar‐specific effects: The proportion of 1st instars feeding nearest the leaf petiole decreased as these individuals moved to adjacent leaves, while the proportion of 2nd–3rd instars feeding nearest the leaf petiole remained constant. Fourth instars also dispersed to neighboring leaves after pheromone exposure. Lastly, alarm pheromone reception caused maternal aphids to alter their preferred feeding sites in a genotype‐specific manner: Maternal aphids of the green genotype fed further from the petiole, while maternal aphids of the pink genotype fed closer to the petiole. Together, our results suggest that aphid colony responses to alarm pheromone constitute a diversity of nuanced instar‐ and genotype‐specific effects. These behavioral responses can dramatically change the spatial organization of colonies and their collective defensive behavior.     

Is the response of aphids to alarm pheromone stable?

Aphid taxa are characterized by a number of biological features, such as their feeding behaviour and host selection, which it is generally accepted are affected by keeping them for several generations under standard conditions in a laboratory. Analyses of three strains of the green pea aphid, Acyrthosiphon pisum (Harris, 1776), reared in culture for long periods, indicate that other characters are also affected. For example, the response of these aphids to alarm pheromone is dramatically reduced. This raises an interesting question regarding the mechanism by which it occurs and has consequences when aphids from laboratory cultures are used for studies in ecology and applied biology and especially the long‐term effectiveness of crop plants genetically engineered to produce EBF as a means of controlling aphids.     

Adjusting the scent ratio: using genetically modified Vitis vinifera plants to manipulate European grapevine moth behaviour

Herbivorous insects use olfactory cues to locate their host plant within a complex olfactory landscape. One such example is the European grapevine moth Lobesia botrana, a key pest of the grape in the Palearctic region, which recently expanded both its geographical and host plant range. Previous studies have showed that a synthetic blend of the three terpenoids, (E)‐β‐caryophyllene, (E)‐β‐farnesene and (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT), was as attractive for the moth as the complete grape odour profile in laboratory conditions. The same studies also showed that the specific ratio of these compounds in the grape bouquet was crucial because a percentage variation in any of the three volatiles resulted in almost complete inhibition of the blend's attractiveness. Here, we report on the creation of stable grapevine transgenic lines, with modified (E)‐β‐caryophyllene and (E)‐β‐farnesene emission and thus with an altered ratio compared to the original plants. When headspace collections from these plants were tested in wind tunnel behavioural assays, they were less attractive than control extracts. This result was confirmed by testing synthetic blends imitating the ratio found on natural and transformed plants, as well as by testing the plants themselves. With this evidence, we suggest that a strategy based on volatile ratio modification may also interfere with the host‐finding behaviour of L. botrana in the field, creating avenues for new pest control methods.     

Combining pheromone and kairomones for effective trapping of the pine sawyer beetle Monochamus galloprovincialis

Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae) is a secondary wood borer that acquired primordial importance since it was identified as the European vector of Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD). An effective trapping method is needed as a tool for managing this insect vector and allowing early detection of nematode transportation. Among effective attractants identified in recent years are the specific M. galloprovincialis aggregation pheromone, host pine kairomones such as α‐pinene and bark beetle kairomones like ipsenol and methyl‐butenol. The main objective of this study was to optimize the combination of these volatiles to improve lure attractiveness and specificity. Based on ten complementary field experiments, we found a pheromone dose‐response of trap catches. The best combination of attractants was the aggregation pheromone plus two bark beetle kairomones, ipsenol and methyl‐butenol. Addition of pine terpenes, such as α‐pinene, did not significantly improve M. galloprovincialis trap capture, but did increase catch of non target species, including natural enemies. The use of pine terpenes would be advisable only if priorizing to maximize removal of vectors. While this research has lead to the development a new, highly attractive commercial lure for mature pine sawyers, none of the tested blends were successful in attracting immature pine sawyer adults. Further investigation is needed to develop attractants for these beetles.     

Is there any evidence that aphid alarm pheromones work as prey and host finding kairomones for natural enemies?

1. The aphid alarm pheromone (E)‐β‐farnesene (EBF) is often considered to be used by natural enemies as a prey/host finding kairomone. However, studies show opposing results, some appear to confirm an attraction of aphid natural enemies by EBF whereas others do not provide any evidence for the kairomone function of EBF. 2. To clarify if aphid natural enemies are attracted by the amounts of EBF naturally emitted by aphids, the existing literature was reviewed about EBF attractiveness to aphid natural enemies with consideration of the amounts of EBF used in the studies. 3. Thirty‐one publications that investigated the ability of EBF, aphid cornicle secretion, and attacked aphids, to attract aphid natural enemies were found. Several studies showed an attraction by EBF, but these used much higher amounts of EBF than usually emitted by aphids during a predator attack. Studies investigating EBF amounts similar to what is emitted by aphids are rare and failed to show attraction. Only two studies document an attraction of natural enemies by attacked aphids. 4. As EBF is emitted in very low amounts, not very stable, and only present after an attack, we suggest that aphid‐derived EBF is not a suitable kairomone for most natural enemy species, especially when they are able to use alternative cues. As EBF, amongst other volatiles, is also emitted by herbivore‐induced plants, we propose that natural enemies might use plant‐derived EBF as a synomone to identify aphid‐infested plants via an altered plant volatile bouquet.     

Amino acid chalcogen analogues as tools in peptide and protein research

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur‐containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl‐tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl‐ and phenylalanyl‐tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl‐tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.     

Field response of the northern spruce bark beetle Ips duplicatus (Sahlberg) (Coleoptera: Curculionidae,Scolytinae) to different combinations of synthetic pheromone with (−)‐α‐pinene and (+)‐limonene

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Acetate formation during recombinant protein production in Escherichia coli K‐12 with an elevated NAD(H) pool

Acetate formation is a disadvantage in the use of Escherichia coli for recombinant protein production, and many studies have focused on optimizing fermentation processes or altering metabolism to eliminate acetate accumulation. In this study, E. coli MEC697 (MG1655 nadR nudC mazG) maintained a larger pool of NAD(H) compared to the wild‐type control, and also accumulated lower concentrations of acetate when grown in batch culture on glucose. In steady‐state cultures, the elevated total NAD(H) found in MEC697 delayed the threshold dilution rate for acetate formation to a growth rate of 0.27 h^?1. Batch and fed‐batch processes using MEC697 were examined for the production of β‐galactosidase as a model recombinant protein. Fed‐batch culture of MEC697/pTrc99A‐lacZ compared to MG1655/pTrc99A‐lacZ at a growth rate of 0.22 h^?1 showed only a modest increase of protein formation. However, 1 L batch growth of MEC697/pTrc99A‐lacZ resulted in 50% lower acetate formation compared to MG1655/pTrc99A‐lacZ and a two‐fold increase in recombinant protein production.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Quantification of ventricular β2‐adrenoceptor density and ligand binding affinity in wild sockeye salmon Oncorhynchus nerka smolts using a novel modification to the tritiated ligand technique

A new, image‐based, tritiated ligand technique for measuring cardiac β₂‐adrenoceptor (β₂‐AR) binding characteristics was developed and validated with adult rainbow trout Oncorhynchus mykiss hearts so that the tissue limitation of traditional receptor binding techniques could be overcome and measurements could be made in hearts nearly 14‐times smaller than previously used. The myocardial cell‐surface (functional) β₂‐AR density of O. nerka smolts sampled at the headwaters of the Chilko River was 54·2 fmol mg protein^?1 and about half of that previously found in return migrating adults of the same population, but still more than twice that of adult hatchery O. mykiss (21·1 fmol mg protein^?1). This technique now opens the possibility of investigating cardiac receptor density in a much wider range of fish species and life stages.     

Mitofusin‐2 knockdown increases ER–mitochondria contact and decreases amyloid β‐peptide production

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca²⁺ transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca²⁺ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ₄₀ and Aβ₄₂. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.     

Attracting Common Carp to a bait site with food reveals strong positive relationships between fish density,feeding activity,environmental DNA,and sex pheromone release that could be used in invasive fish management

Measurement of environmental DNA (eDNA) is becoming a common technique to survey for rare and invasive fish due to its sensitivity and specificity. However, its utility is limited by an incomplete understanding of factors governing its sources and fates. Failure to detect eDNA is especially difficult to interpret so surveillance techniques often collect large numbers of samples across broad regions. If, however, fish could be reliably attracted to a single location where their eDNA could be easily measured that would be useful. We conducted a proof‐of‐concept study of this idea using invasive Common Carp. We monitored the distribution of radio‐tagged Carp and their eDNA across a 67 ha lake focusing at the bait site while a pheromone (Prostaglandin F_2α; PGF_2α) was also measured to determine their reproductive condition. Prior to baiting, Carp were patchily distributed and while eDNA was occasionally detectable, it was patchy and only loosely associated with moderately dense groups of fish. Further, neither Carp, nor their eDNA were consistently measurable at the bait site and surrounding region, and the pheromone was not measurable at all. However, once baiting commenced, Carp started visiting the bait site and feeding, especially at night, where eDNA levels increased 500‐fold as fish densities doubled and PGF_2α became detectable. Fish presence, eDNA and pheromone concentrations peaked at night after 6 days, strongly suggesting feeding activity was the main driver. While the presence of eDNA precisely coincided with this aggregation, levels had dropped dramatically within 5 m. PGF_2α levels dropped less rapidly and demonstrated the presence of live mature fish. We suggest that food could be used to train fish to come to locations where they otherwise are too scarce to be reliably measured, increasing their eDNA release, making them measurable, and their reproductive condition also discernable by measuring pheromones.     

Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.     

The role of the C(2) configuration and methyl substitution on the catalytic activity of novel 2,3,3‐ and 2,7,7‐trimethyl‐substituted γ‐aminonorbornan‐2‐ols

A new set of optically active 2,3,3‐ and 2,7,7‐trimethyl‐substituted γ‐aminonorbornan‐2‐ols have been obtained from 2‐methylenenorbornane‐1‐carbonitriles derived from (+)‐camphor and (?)‐fenchone and probed as chiral ligands for the enantioselective addition of diethylzinc to benzaldehyde. This has allowed the study of the structural factors influencing the chirality transfer, such as variation of the relative configuration at C(2) and steric hindrance at C(2), C(3), and C(7) positions of norbornane, which result in the observance of the important role played by the gem‐dimethyl position in γ‐aminonorbornan‐2‐exo‐ols. An empirical rationalization of the obtained experimental results has been realized on the basis of energetically‐favored diastereomeric Noyori‐like transition states. Chirality 2010. © 2010 Wiley‐Liss, Inc.