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1.  Vertical T-maze Choice Assay for Arthropod Response to Odorants 
Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.
doi:10.3791/50229
PMCID: PMC3601206  PMID: 23439130
Biochemistry; Issue 72; Molecular Biology; Basic Protocols; Entomology; Behavior; Eukaryota; Organic Chemicals; Chemical Actions and Uses; Life Sciences (General); Behavioral Sciences; Arthropod behavior; chemical ecology; olfactometer; chemotaxis; olfaction; attraction; repulsion; odorant; T-maze; psyllid;  Diaphorina citri; insect; anthropod; insect model
2.  Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen 
PLoS Pathogens  2012;8(3):e1002610.
Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.
Author Summary
In this investigation, we experimentally demonstrate specific mechanisms through which a bacterial plant pathogen induces plant responses that modify behavior of its insect vector. Candidatus Liberibacter asiaticus, a fastidious, phloem-limited bacterium responsible for causing huanglongbing disease of citrus, induced release of a specific volatile chemical, methyl salicylate, which increased attractiveness of infected plants to its insect vector, Diaphorina citri, and caused vectors to initially prefer infected plants. However, the insect vectors subsequently dispersed to non-infected plants as their preferred location of prolonged settling because of likely sub-optimal nutritional content of infected plants. The duration of initial feeding on infected plants was sufficiently long for the vectors to acquire the pathogen before they dispersed to non-infected plants, suggesting that the bacterial pathogen manipulates behavior of its insect vector to promote its own proliferation. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen and was similar under both light and dark conditions. Feeding on citrus by D. citri adults also induced the release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants.
doi:10.1371/journal.ppat.1002610
PMCID: PMC3310815  PMID: 22457628
3.  Sexual Transmission of a Plant Pathogenic Bacterium, Candidatus Liberibacter asiaticus, between Conspecific Insect Vectors during Mating 
PLoS ONE  2011;6(12):e29197.
Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 µm in length and 0.19 to 0.39 µm in width. The spherical structures measured from 0.61 to 0.80 µm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees.
doi:10.1371/journal.pone.0029197
PMCID: PMC3244449  PMID: 22216209
4.  Towards crystal structure prediction of complex organic compounds – a report on the fifth blind test 
Following on from the success of the previous crystal structure prediction blind tests (CSP1999, CSP2001, CSP2004 and CSP2007), a fifth such collaborative project (CSP2010) was organized at the Cambridge Crystallographic Data Centre. A range of methodologies was used by the participating groups in order to evaluate the ability of the current computational methods to predict the crystal structures of the six organic molecules chosen as targets for this blind test. The first four targets, two rigid molecules, one semi-flexible molecule and a 1:1 salt, matched the criteria for the targets from CSP2007, while the last two targets belonged to two new challenging categories – a larger, much more flexible molecule and a hydrate with more than one polymorph. Each group submitted three predictions for each target it attempted. There was at least one successful prediction for each target, and two groups were able to successfully predict the structure of the large flexible molecule as their first place submission. The results show that while not as many groups successfully predicted the structures of the three smallest molecules as in CSP2007, there is now evidence that methodologies such as dispersion-corrected density functional theory (DFT-D) are able to reliably do so. The results also highlight the many challenges posed by more complex systems and show that there are still issues to be overcome.
doi:10.1107/S0108768111042868
PMCID: PMC3222142  PMID: 22101543
5.  Towards crystal structure prediction of complex organic compounds – a report on the fifth blind test 
The results of the fifth blind test of crystal structure prediction, which show important success with more challenging large and flexible molecules, are presented and discussed.
Following on from the success of the previous crystal structure prediction blind tests (CSP1999, CSP2001, CSP2004 and CSP2007), a fifth such collaborative project (CSP2010) was organized at the Cambridge Crystallographic Data Centre. A range of methodologies was used by the participating groups in order to evaluate the ability of the current computational methods to predict the crystal structures of the six organic molecules chosen as targets for this blind test. The first four targets, two rigid molecules, one semi-flexible molecule and a 1:1 salt, matched the criteria for the targets from CSP2007, while the last two targets belonged to two new challenging categories – a larger, much more flexible molecule and a hydrate with more than one polymorph. Each group submitted three predictions for each target it attempted. There was at least one successful prediction for each target, and two groups were able to successfully predict the structure of the large flexible molecule as their first place submission. The results show that while not as many groups successfully predicted the structures of the three smallest molecules as in CSP2007, there is now evidence that methodologies such as dispersion-corrected density functional theory (DFT-D) are able to reliably do so. The results also highlight the many challenges posed by more complex systems and show that there are still issues to be overcome.
doi:10.1107/S0108768111042868
PMCID: PMC3222142  PMID: 22101543
prediction; blind test; polymorph; crystal structure prediction

Results 1-5 (5)