Ecdysis period of Rhodnius prolixus head investigated using phase contrast synchrotron microtomography

Highlights

• SR-PhC-μCT were performed using the new set-up available at LNLS – Brazil.

• 3D sections of Rhodnius prolixus head were obtained using SR-PhC-μCT.

• Results showed structures of R. prolixus in the ecdysis period.

Abstract

Microtomography using synchrotron sources is a useful tool in biological imaging research since the phase coherence of synchrotron beams can be exploited to obtain images with high contrast resolution. This work is part of a series of works using phase contrast synchrotron microtomography in the study of Rhodnius prolixus head, the insect vector of Chagas’ disease, responsible for about 12,000 deaths per year. The control of insect vector is the most efficient method to prevent this disease and studies have shown that the use of triflumuron, a chitin synthesis inhibitor, disrupted chitin synthesis during larval development and it’s an alternative method against insect pests.

The aim of this work was to investigate the biological effects of treatments with triflumuron in the ecdysis period (the moulting of the R. prolixus cuticle) using the new imaging beamline IMX at LNLS (Brazilian Synchrotron Light Laboratory). Nymphs of R. prolixus were taken from the Laboratory of Biochemistry and Physiology of Insects, Oswaldo Cruz Foundation, Brazil. Doses of 0.05 mg of triflumuron were applied directly to the abdomen on half of the insects immediately after feeding. The insects were sacrificed 25 days after feeding (intermoulting period) and fixed with glutaraldehyde.

The results obtained using phase contrast synchrotron microtomography in R. prolixus showed amazing images of the effects of triflumuron on insects in the ecdysis period, and the formation of the new cuticle on those which were not treated with triflumuron. Both formation and malformation of this insect’s cuticle have never been seen before with this technique.

Introduction

Absorption contrast was, for a long time, the only contrast mechanism that could be used for the X-ray imaging and reconstruction of 3D structures. However this technique was only effective on highly absorbing samples, which presented either a high X-ray attenuation coefficient or consisted of components with sufficiently large differences in their attenuation coefficients [1].

Recent years have experienced a growing interest in various forms of synchrotron radiation phase-contrast microtomography in order to visualize materials with low densities [2], [3]. Phase contrast is caused by modification of the geometry of a planar wave front and the arrival of this technique made possible the non-destructive investigation of materials with low X-ray attenuation coefficients such as insects and biological samples, enhancing mainly the visibility of borders and soft tissues in the images [4], [5]. Synchrotron radiation phase-contrast microtomography is often particularly sensitive to high spatial frequency features, giving an alternative view of the sample and being useful for investigating microstructure inside insects and biological specimens, without staining them with a contrast medium [2].

In this work, we report on the particular advantages of synchrotron radiation phase-contrast microtomography, using the new imaging beamline IMX at LNLS (Brazilian Synchrotron Light Laboratory), in the study of the biological effects of treatments with triflumuron (TFM) (Starycide® sc 480 Bayer) on the fifth instar nymph of Rhodnius prolixus, the most important insect vector of Chagas’ disease.

Chagas’ disease, also known as a American trypanosomiasis, is a widely spread illness in South America disseminated by hematophagous insects and causes about 12,000 deaths per year. From thirty to forty percent of the infected people develop cardiomyopathy and/or digestive syndromes [6].

The blood-sucking bug R. prolixus undergoes five nymphal stages. At the fifth moulting, when the insect becomes adult, there are striking changes in all parts of the body. Moulting in R. prolixus occurs at a definite interval after feeding, only one meal being necessary to trigger the process in each stage. The moulting to the fifth nymph usually happens about 25 days after feeding [7].

The primary mode of action of treatments with triflumuron (TFM) (Starycide® sc 480 Bayer) is to disrupt chitin synthesis during larval development, which is the period that determines moulting inhibition and malformations of the cuticle. Chitin is a linear polymer of N-acetylglucosamine, an essential element of the exoskeleton in insects. During ecdysis (the shedding or casting off of an outer coat), chitin metabolism is increased to produce a new exoskeleton. The old exoskeleton is resorbed, and parts of the digested material will be recycled [8].

Nowadays, the chemical insecticide constitutes the most important tool for vector population control programs, although several of them also induced resistance on the target insects. Thus, data suggest that TFM and perhaps other chitin synthesis inhibitors may be considered as potential tools for integrated vector control programs against hematophagous triatomine species [9].

First works [10], [11], [12], [13], [14], [15] using synchrotron radiation phase-contrast microtomography in R. prolixus showed that this technique is an excellent tool to visualize the internal and external structures of R. prolixus.

The high intensity and high transverse coherence of delivered radiation from last generation synchrotron sources provides best qualities in comparison with other sources: orders of magnitude higher on photon flux density; the almost parallel beam propagation; the coherence properties of the beam arriving at the sample; a significantly better resolution; a larger signal-to-noise ratio; and short acquisition times. These characteristics, together with the development of novel X-ray detectors and faster computers, have increased the interest in the use of microtomography applied to biological samples, as they allow an improvement in resolution and the use of propagation phase contrast (due to the parallel beam and high flux, the distance between source and experiment can be extended up to several centimeters) [1], [16], [17].

Propagation-based phase contrast involves placing the detector some distance from the sample, so that the radiation refracted from the sample can interfere with the unchanged beam [18]. Phase-sensitive μCT is characterized by an enhanced contrast in regions of highly localized change in refractive index, such as sample borders or interfaces between the original object and included materials [19].

In this paper, we use phase contrast synchrotron microtomography at IMX beamline to investigate the behavior of internal structures of R. prolixus treated with TFM in the intermoulting period and compared the results with a control group of R. prolixus.