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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Streszczenie

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Wprowadzenie

Compounds like DEET, Picaridin, Citronellal and IR3535 have been shown to effectively repel mosquitoes, including the important disease vector Aedes aegypti1,2. We record action potentials from sensory neurons associated with specific gustatory sensilla to determine the cells involved with mosquito repellency. Coupled with downstream sequencing of expressed genes in these tissues, we may identify the genes most likely mediating the responses of these cells in order to screen new compounds for improved repellency capabilities.

RNA-seq is a powerful tool, quickly becoming standard for tracking temporal and spatial changes ....

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Protokół

1. Rearing Ae. aegypti adults

  1. Hatch eggs in approximately ¾ inch water in shallow tray. Overcrowding will reduce the size of adults.
  2. Rear larvae at 25 °C (12-hL:12-hD) and feed with ground fish food.
    NOTE: Overfeeding may reduce survival rates.
  3. Remove pupae individually by Pasteur pipette daily and transfer to plastic dishes (9 cm × 5.5 cm) inside small containment buckets with fine mesh lids, thus establishing 24 hr age groupings.
  4. Feed adult mosquitoes 10% sucrose solution by cotton ball placed on the screen walls of a cage housing the mosquitoes in an environmental chamber at 27 °C an....

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Wyniki

The trace recordings of action potentials from Ae. aegypti gustatory sensilla (Figure 1) demonstrate the effectiveness of direct stimulation with a range of chemicals. This technique can be used to quantify responses to any stimulating chemical by counting spikes of a given amplitude and duration over a reasonable time range (generally less than 500 ms). Trace recordings must be readily reproducible under a given set of experimental conditions. Otherwise, the observed physiological responses may.......

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Dyskusje

The most challenging aspect of recording action potentials from gustatory sensilla is deciding which responses are “normal.” When employing single gustatory sensillum tip recording the first time for a given insect species, the total number and sensitivities of the gustatory receptor neurons (GRNs) are likely unknown. Many preliminary recordings are first required to decide the range and concentrations of chemicals to test. In this instance, we began with the observation that a single GRN responded to low con.......

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

The authors thank Bryan T. Vinyard of the USDA, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Biometrical Consulting Service, Beltsville, MD for statistical analyses. This work was supported in part by a grant to J.C.D. from the Deployed War Fighter Protection (DWFP) Research Program funded by the Department of Defense through the Armed Forces Pest Management Board (AFPMB).

....

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Materiały

NameCompanyCatalog NumberComments
Glass capillaryA-M Systems628000Standard, 1.5 mm X 0.86 mm, 4"
Silver wireA-M Systems7875.015" bare
Tungsten wireAlfa Products3690.127 mm diameter
Fine forcepsFine Science Tools11252#5SF Inox
Refridgerated stageBioQuip Products1429Chill Table
PreamplifierSyntechTaste Probepreamplifier
Software for electrophysiologySyntechAutospikesoftware for electrophysiology
TetraMin fish foodTetraTropical fish food granulesfish food ground to fine powder
TRIzolLife Technologies15596-026RNA isolation reagent
RNeasy Plus Mini KitQiagen74136includes gDNA eliminator and RNeasy spin columns
Nanodrop spectrophotometerNano Drop ProductsND-1000tabletop spectrometer
R statisticsfreeware (created by Robert Gentleman and Ross Ihaka)www.r-project.orgUse the lm function of the stats package and the equiv.boot function of the equivalence package in the R computing environment.
1.5 ml tube rackEvergreen240-6388-030Pour liquid nitrogen into a few empty wells to freeze and grind tissue.
1.5 ml collection tubes with pestleGrainger6HAX6RNase free
CentrifugeThermo Scientific11178160Spin down frozen tissue to keep at bottom of 1.5 ml tube.
Primer-BLAST Primer Designing toolNCBIn/a

Odniesienia

  1. Klun, J. A., Khrimian, A., Debboun, M. Repellent and deterrent effects of SS220, picaridin, and DEET suppress human blood feeding by Aedes aegypti, Anopheles stephensi, and Phlebotomus papatasi. J. Med. Entomol. 43 (1), 34-39 (2006).
  2. Dickens, J. C., Bohbot, J. D.

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Keywords Aedes AegyptiMosquitoGustatory AppendagesElectrophysiologyRNA SequencingChemoreceptionLabellumTarsiRepellent PerceptionDEETQuinine

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