Following de novo triglyceride dynamics in ovaries of Aedes aegypti during the previtellogenic stage

Abstract

Understanding the molecular and biochemical basis of egg development is a central topic in mosquito reproductive biology. Lipids are a major source of energy and building blocks for the developing ovarian follicles. Ultra-High Resolution Mass Spectrometry (UHRMS) combined with in vivo metabolic labeling of follicle lipids with deuterated water (²H₂O) can provide unequivocal identification of de novo lipid species during ovarian development. In the present study, we followed de novo triglyceride (TG) dynamics during the ovarian previtellogenic (PVG) stage (2-7 days post-eclosion) of female adult Aedes aegypti. The incorporation of stable isotopes from the diet was evaluated using liquid chromatography (LC) in tandem with the high accuracy (< 0.3 ppm) and high mass resolution (over 1 M) of a 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (14.5 T FT-ICR MS) equipped with hexapolar detection. LC-UHRMS provides effective lipid class separation and chemical formula identification based on the isotopic fine structure. The monitoring of stable isotope incorporation into de novo incorporated TGs suggests that ovarian lipids are consumed or recycled during the PVG stage, with variable time dynamics. These results provide further evidence of the complexity of the molecular mechanism of follicular lipid dynamics during oogenesis in mosquitoes.

Figure 1

Deuterium incorporation into triglycerides occurs during de novo fatty acid synthesis. Different tissue compartments are represented (crop, midgut, hemolymph, ovary and fat body). The TG synthetic pathway utilizes substrates that have been labelled with deuterium, including acetyl CoA, NADPH and water. Hydrogen atoms (H) highlighted in red indicate the location where deuterium (D) may have replaced hydrogen in a newly synthesized molecule. TCA cycle tricarboxylic acid cycle, CoASH coenzyme A, RCO-S-CoA acetyl-coenzyme A, malonyl-CoA malonyl coenzyme A, NADPH nicotinamide adenine dinucleotide phosphate, LTP lipid transfer particle, LP lipophorin, DG diglyceride, TG triglyceride.

Figure 2

Typical extracted ion chromatograms of the [M + NH₄]⁺ molecular ion form for all the TG species observed. In the insets, typical MS projection showing the relative abundance of the [M + NH₄]⁺ and [M + Na]⁺ molecular ion forms.

Figure 3

(A,B) Typical MS projections for the signal corresponding to 48:2 TG from ovarian samples. Deuterium labeled diet (A) and normal diets (B). Note the increase complexity of the MS signals with increases of the number of deuteriums incorporated and ion forms. (C,D) Amplified MS projections denoting the sources for isobaric interferences in the 820–834 m/z range, and the corresponding nominal masses. The TG species containing deuterium are highlighted in red.

Figure 4

Total TG per ovary as a function of diet and time. Upper panel: 20% Sucrose/Heavy water diet. The numbers in the top are referring to the percentages of ²H incorporated into TGs. Lower panel: 20% sucrose/HPLC water diet. Bars are means standard error (± SE) of the analysis of triplicates samples. Each sample contained 10 ovaries.

Figure 5

Distribution of the number of deuteriums per TG species as a function of days after eclosion (2–7 days). The median number of deuteriums is shown in the insets (white label). The color scale corresponds to the amount of deuterated species relative to the TG 48:1 (d7) internal standard.