Coherent anti-Stokes Raman scattering (CARS) spectroscopy in Caenorhabditis elegans and Globodera pallida: evidence for an ivermectin-activated decrease in lipid stores

Abstract

Background: Macrocyclic lactones are arguably the most successful chemical class with efficacy against parasitic nematodes. Here we investigated the effect of the macrocyclic lactone ivermectin on lipid homeostasis in the plant parasitic nematode Globodera pallida and provide new insight into its mode of action.

Results: A non-invasive, non-destructive, label-free and chemically selective technique called Coherent anti-Stokes Raman scattering (CARS) spectroscopy was used to study lipid stores in G. pallida. We optimised the protocol using the free-living nematode Caenorhabditis elegans and then used CARS to quantify lipid stores in the pre-parasitic, non-feeding J2 stage of G. pallida. This revealed a concentration of lipid stores in the posterior region of J2 s within 24 h of hatching which decreased to undetectable levels over the course of 28 days. We tested the effect of ivermectin on J2 viability and lipid stores. Within 24 h, ivermectin paralysed J2 s. Counterintuitively, over the same time-course ivermectin increased the rate of depletion of J2 lipid, suggesting that in ivermectin-treated J2 s there is a disconnection between the energy requirements for motility and metabolic rate. This decrease in lipid stores would be predicted to negatively impact on J2 infective potential.

Conclusion: These data suggest that the benefit of macrocyclic lactones as seed treatments may be underpinned by a multilevel effect involving both neuromuscular inhibition and acceleration of lipid metabolism. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Keywords: Caenorhabditis elegans; Raman spectroscopy; Sudan Black; abamectin; metabolism; nematicide; potato cyst nematode; seed treatment; starvation.

Figure 1

A Coherent anti‐Stokes Raman scattering (CARS) spectrum from Caenorhabditis elegans. This shows that the peak is at ∼2850 cm^‐1. This corresponds well to the vibrational frequency expected for neutral lipids in the C‐H stretching region and provides validation of this approach for detection of lipid.

Figure 2

Representative CARS images of different regions, (a) head, (b) abdomen and (c) tail, in 1‐day‐old adult C. elegans. The corresponding segments are shown in the schematic below the images. CARS images were obtained by tuning to the ‐CH₂ stretching frequency at 2845 cm^‐1 for lipids. Lipid‐rich areas (lipid stores) appear as bright red/yellow puncta in the images, as indicated by the arrows. The image shown is representative of 10 similar images taken for each time‐point.

Figure 3

Sudan Black staining of wild‐type 1‐day‐old adult C. elegans worms which were either well‐fed or starved. (a − d) Photographs of well‐fed worms (a, c) and worms that had been food‐deprived for 24 h (b, d). Red arrows indicate the Sudan Black staining. (e) Sudan Black staining in C. elegans at different stages during food deprivation. The intensity was measured by highlighting the stained area as a region of interest. Pixels in a specified area were counted as the intensity. Every population of food‐deprived worms (open bars) was paired with a control group (hatched bars). During the first 5 h of food deprivation, there was no reduction in Sudan Black staining. After 10 h in the absence of food, there was a significant reduction in the level of Sudan Black staining and an even further decrease after 24 h of food withdrawal (n ≥ 10; mean ± standard error of the mean; ***P < 0.001).

Figure 4

CARS images of regions of interest (a, head; b, middle; c, posterior/tail) from C. elegans after different times without food. The area of the worm from which images were collected is indicated in the left panels accompanied by representative images of the CARS signal (bright puncta correspond to the lipid vibrational frequency). Images from left to right were collected at increasing periods of food deprivation (0.5 to 24 h) and over this time‐course the abundance of the bright signal, i.e. the lipid vibrational frequency, decreases. Values have been normalized to those of control samples (well‐fed worms) and averaged from two replicates, each with n = 3. ****P < 0.0001; ***P < 0.001; **P < 0.01, and *P < 0.05).

Figure 5

CARS images of G. pallida J2s. (a) The regions of the worm that were imaged; unshaded is the anterior/head region and shaded is the posterior/tail region. (b) The CARS signal corresponding to the lipid vibrational frequency from the anterior/head region is very weak. (c) In contrast, the CARS signal from the posterior/tail region is strong, as indicated by the extensive bright yellow/red regions.

Figure 6

(a) Representative CARS images of the posterior/tail region of Globodera pallida J2s after hatching. The number of days after hatching is indicated on each image. The CARS signal corresponding to the lipid vibrational frequency (bright yellow/red areas) is depleted over time post‐hatching. (b) Quantitative analysis of lipid stores from CARS images showed a decrease after hatching in J2 G. pallida. Values have been normalized to values corresponding to 1‐day‐old worms and averaged from two independent data sets, each with n = 10. ****P < 0.0001; ***P < 0.001; **P < 0.01, and *P < 0.05.

Figure 7

Ivermectin inhibits the motility of G. pallida. (a) Ivermectin inhibited dispersal of J2s from the centre, ‘origin’, of an agar plate in a concentration‐dependent manner after 2 h of exposure. Data are mean ± standard error of the mean. The experiment was repeated six times on two different days. Vehicle for the ivermectin was 0.5% ethanol, and 0.5% ethanol was incorporated in the control plate. (b) Two hours of exposure to 10 µM ivermectin causes a marked flaccid paralysis, characterised by the loss of postural shape of the worms. Each image is representative of 10 samples.

Figure 8

CARS analysis of J2 G. pallida incubated with ivermectin. J2s were selected within 1 day of hatching and incubated with 1 µM ivermectin or vehicle (control) for 24 h. CARS signals corresponding to the lipid vibrational frequency were measured from the head, middle and posterior region of each worm. The signals from the ivermectin‐treated worms were normalised with respect to the vehicle control. n = 3. ***P < 0.001.