Assessment of biophysical properties of Haemonchus contortus from different life cycle stages with atomic force microscopy
Graphical abstract
Introduction
The wavelength limit of the visible electromagnetic radiation in the optical microscopes motivated the development of advanced non-light based microscopies. These new techniques of microscopy enabled the investigation of several systems in scales that reach the subnanometric world. Atomic Force Microscopy (AFM) is a high resolution microscopy technique that provides information about the topography and surface composition of a wide variety of materials ranging from living individual cells [1] through fixed tissue [2]. As the name of the technique suggests, the AFM can image local forces between the surface of the sample and the tip, including van der Walls forces, Born repulsion, electrostatic forces, magnetic forces, friction, adhesion, as well as map structural properties of the samples, such as roughness, viscosity and elasticity, correlating these properties with the structure of the studied material.
Akhatova et al. [3] report that the recent application of the AFM technique in the study of large invertebrates, with challenging three-dimensional image acquisitions, limiting the application of this powerful technique in this type of sample. By this fact, all studies of AFM in nematodes are still very new and challenging.
Nematodes, in general, share a very similar anatomy, morphology and physiology. However, striking differences among the species can be substantial, especially if the worms are from different habitats [3]. Little is known about the biophysical properties of worm parasites [4], and to the best of our knowledge, the present study is the first to show the biophysical properties of several stages of the ruminant worm parasite Haemonchus contortus.
H. contortus is the most pathogenic and economically important gastrointestinal nematode species infecting sheep and goats worldwide [5]. These parasites cause severe anemia, weight loss and death in some cases. The greatly increased prevalence of gastrointestinal nematode resistance to all available anthelmintics [6] leads to a critical need for alternatives as new compounds or nonchemical control methods as well as tools that allow for the evaluation of the effects or mechanisms of action of gastrointestinal nematodes.
Light, scanning and transmission electron microscopy have been successfully used to assess damage or structural parameters of H. contortus in different life cycle stages in several in vitro or in vivo studies [7], [8], [9], [10]. However, some limitations of these techniques are that they limit advances in understanding the effect of different compounds on H. contortus or the analyses of this parasite in different biochemical and physiological situations. For the free live nematode Caenorhabditis elegans and the human hookworm Necator americanus, important surface parameters were discovered with atomic force microscopy (AFM) experiments, which could not be detected using transmission and scanning electron microscopies, despite providing high-resolution images [4], [11]. The conventional sample preparation methodology or working principle for electron microscopies (transmission electron microscopy and scanning electron microscopy), such as metal coating and high energies of electron beams, may damage or alter nematode cuticle structures.
AFM has demonstrated its power in applications ranging from studies on pure materials to studies on biomedicine with informative morphological and mechanical properties of biological samples [12]. Although AFM is a versatile and established tool to study the surface properties of biological specimens [13], important surface parameters of only a few nematode species have been successfully characterized by AFM [11], [14] and, to the best of our knowledge, there are no studies with ruminant nematode parasites such as H. contortus cuticles or analyses of different H. contortus stages using AFM.
Egg and larvae stages of H. contortus (Fig. 1) are found outside of their host animals, whereas adult stages live inside their warm-blooded hosts [15]. In the abomasum of the ruminant host, the H. contortus adult females release eggs via the feces and begin the free-living stages [16]. To provide unique data on the H. contortus ultrastructure and biomechanical (adhesion and stiffness) properties that play key roles in some developmental stages of this nematode specimen, the present study report, for the first time, assessed the topographic and biomechanical characterization of H. contortus eggs, larvae and adult cuticle by AFM, revealing structures never seen before with other microscopy techniques.
Section snippets
Haemonchus contortus strain
A monospecific strain of H. contortus was maintained in experimentally infected sheep that received hay and water ad libitum, and 1% of its live weight of commercial food was 20% crude protein. All procedures were approved through the Ethics Committee on Animal Experimentation of Federal University of Maranhão, Brazil, under protocol number 23115.005443/2017-51.
Acquisition and fixation of Haemonchus contortus eggs and larvae
H. contortus eggs were obtained from fresh feces from sheep that were experimentally infected according to Coles et al. [17]. The feces
Results
AFM was used to obtain topographic images of different stages of H. contortus. Eggs in the morulae and larvae stages present differences in terms of their surface structures, such as roughness and height features (Fig. 2). The mean of Rrms roughness values calculated for morulae and larvae egg stages were 234.50 ± 82.43 nm and 79.25 ± 11.50 nm, respectively (the results are shown as the mean ± standard deviation). The statistical t-test applied to roughness data showed that morulae eggs
Discussion
The nematode egg is an important stage of the parasite’s life cycle, and it may be a potential target for control strategies. The eggshell is a complex structure that includes three layers as follows: an external vitelline, a medial chitinous layer and a basal lipid/protein layer [28]. In this context, the important observation is that the embryonation of eggs increased resistance to adverse environmental conditions and anthelmintics after natural modification of eggshells and changes in
Conclusions
We employed AFM characterization to provide morphological and biophysical information of surfaces of the H. contortus nematode in different life stages. Our results provide early insight into the differential biomechanical and ultrastructural properties of our samples, which can explain biological and biochemical steps in the life cycle of these parasites and is also important to open possibilities to investigate new approaches to control nematodes infections.
Declaration of Competing Interest
The authors declare that there is conflict of interest regarding the publication of this article.
Acknowledgments
This research was supported by IECT Biotechnology through grants from FAPEMA (Maranhão State Research Foundation, Brazil) and FINEP (Brazilian Innovation Agency). C.R. Silva received a postgraduate scholarship from CAPES (Coordination for the Improvement of Higher Education Personnel, Brazil). We thank Moacir Rogrigues Brasil for graphical support in the elaboration of panels. The authors also thank CNPq (the Brazilian National Council for Scientific and Technological Development) for a
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