Research Article: Using scanning electron microscopy (SEM) to study morphology and morphometry of the isolated haptoral sclerites of three distinct diplozoid species

Date Published: February 25, 2019

Publisher: Public Library of Science

Author(s): Quinton Marco Dos Santos, Ewa Dzika, Annemariè Avenant-Oldewage, Suzannah Rutherford.

http://doi.org/10.1371/journal.pone.0211794

Abstract

Diplozoidae infects the gills of cyprinid fishes in Africa, Europe, and Asia. Traditionally the hardened internal structures, crucial for identification of diplozoid species, are studied using light microscopy. Recently, the sclerotised haptoral structures of an African diplozoid, Paradiplozoon vaalense, were successfully isolated and visualised using scanning electron microscopy (SEM). In this paper, the haptoral sclerites of three diplozoid species are compared using SEM for the first time. Paradiplozoon ichthyoxanthon and Paradiplozoon vaalense occur on Labeobarbus and Labeo species, respectively, in the Vaal River system, South Africa, while Diplozoon paradoxum is widely-distributed in Europe and Asia, infecting several host species. Diplozoon paradoxum is a well-studied species, as well as being the type species of the family and ideal for inclusion in an exploratory study for comparative purposes. SEM study of D. paradoxum and P. ichthyoxanthon provided valuable information regarding surface morphology of the attachment structures hitherto not observed. Elaborate morphometric study of the haptoral hooks were incorporated, adding 12 point-to-point measurements to current morphometric characteristics. The results were analysed statistically, and significant differences support absolute separation (100.00%) of the three species following discriminant analysis. These point-to-point measurements could also be used for light microscopical study in the future and aid species delimitation within the Diplozoidae.

Partial Text

The Monogenea (Platyhelminthes), a widely distributed and highly diverse group of aquatic parasites, affects a substantial range of hosts. Their taxonomy is predominantly based on morphology of hardened structures in their haptoral attachment organs and reproductive systems. These structures, or sclerites, have traditionally been studied using light microscopy techniques of specimens mounted on glass slides. However, accurately describing sclerite from a 2-dimensional perspective, such as that obtained from conventional light microscopy, is limiting, particularly without sectioning and reconstruction. Some of the problems include sclerites not lying perfectly flat or overlapping. Obscured observation of structures situated deep in the parasite tissue, and sub-optimal fixing, mounting, and staining protocols provide further challenges. Maillard et al. [1] successfully released the male copulatory organ (MCO or cirrus) and some of the haptoral sclerites of Diplectanum aequans (Wagener, 1857) from their surrounding tissue using sodium carbonate, allowing these structures to be studied using scanning electron microscopy (SEM). Similar techniques were used to study the sclerites of gyrodactylid monogeneans. Mo and Appleby [2] used artificial gastric juices (HCl and pepsin) to digest the tissue of Gyrodactylus salaris Malmberg, 1957. Shinn et al. [3] combined digestive solutions (HCl–pepsin, 10% KOH, 40% Na2CO3) to release the sclerites of Gyrodactylus species. An inherent flaw of these techniques was the loss of some sclerites due to the differential composition of specific sclerites that resulted in inconsistent resistance to digestion. Disulphide bonds, resistant to pepsin [4] occur within some structures (cirri, hamuli, transverse bars, marginal hooks) and resist digestion to a greater extent than other, such as dorsal and ventral connecting bars. For example, Mo and Appleby [2] also studied sclerites of a larger, clamp-bearing, monogenean, Discocotyle sagittata (Leuckart, 1842), and obtained similar results, with only the marginal hooks being observed after digestion and the clamps being “seen to disappear almost as fast as the soft surrounding tissue” (sic.).

Sclerites constituting the clamps, excluding the small sclerites connecting the anterior end of the median sclerite with the anterior clamp jaws, were successfully retained post-digestion and studied using SEM. Micrographs of the observed structures are presented in Figs 3 to 8. Consistent characteristics across the species studied, as well as recorded data, are provided below. Characters showing variation noted in Table 1.

 

Source:

http://doi.org/10.1371/journal.pone.0211794

 

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