title: Improved techniques for measurement of nanolitre volumes of phloem exudate from aphid stylectomy.
abstract: BACKGROUND
  When conducting aphid stylectomy, measuring accurate rates of phloem exudation is difficult because the volumes collected are in the nanolitre (nl) range. In a new method, exudate volume was calculated from optical measurement of droplet diameter as it forms on the tip of a severed aphid stylet. Evaporation was shown to decrease the accuracy of the measurement but was countered with the addition of water-saturated mineral oil. Volume measurements by optical estimation of the volume of a sphere suspended in oil was affected by the curvature of the oil surface. In contrast, measuring the exudate volume from optical measurement of droplet-diameter as formed on the tip of a severed aphid stylet, removes any inaccuracies due to oil surface curvature. A modified technique is proposed for measuring exudate volumes without oil by estimating the flow rate from photo-sequences of the collection period; a correction for evaporation is applied later.
  
  
  RESULTS
  A change in oil volume of ±1.75% from an optimum volume of 285 μl had a statistically significant effect on droplet measurement, under or over-estimating droplet volume due to optical effects caused by the oil surface. Using microscope image capture and measurement software, a modified method for measuring phloem volume in air was developed, by reducing air exposure during measurement to approximately 5 s for each measurement. Phloem volumes were measured using both techniques with measurements in air being on average 19.9 nl less (SD 18.87, p<0.001) than those made in oil, and there was a strong linear relationship (R(2)=0.942) between the techniques. This linear relationship enabled the development of a correction equation with no significant difference at the 5% level between corrected volumes and actual volumes measured under oil.
  
  
  CONCLUSIONS
  This study showed that oil has a significant role in countering evaporation but oil volume must be carefully optimised for optical measurement of droplets to ensure measurement accuracy. A linear correction factor was generated to correct the volumes measured in air for loss due to evaporation and the method provides for a much simpler alternative to previous approaches for measuring exudation rates and volumes from a cut aphid stylet.
authors.affiliation: School of Biological Science, Flinders University, 5042 Bedford Park, South Australia, Australia.
authors.forename: Lachlan J
authors.initials: LJ
authors.lastname: Palmer
authors.affiliation: School of Agriculture Food and Wine, University of Adelaide, Waite Campus, 5064 Adelaide, South Australia, Australia.
authors.forename: Lyndon T
authors.initials: LT
authors.lastname: Palmer
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, B152TT Birmingham, UK.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Pritchard
authors.affiliation: School of Biological Science, Flinders University, 5042 Bedford Park, South Australia, Australia.
authors.forename: Robin D
authors.initials: RD
authors.lastname: Graham
authors.affiliation: School of Biological Science, Flinders University, 5042 Bedford Park, South Australia, Australia.
authors.forename: James Cr
authors.initials: JC
authors.lastname: Stangoulis
keywords: Aphid stylectomy; Exudate; Phloem; Volume measurement